Di-sulfide containing cell penetrating peptides and methods of making and using thereof

ABSTRACT

Disclosed is a general, reversible bicyclization strategy to increase both the proteolytic stability and cell permeability of peptidyl drugs. A peptide drug is fused with a short cell-penetrating motif and converted into a conformationally constrained bicyclic structure through the formation of a pair of disulfide bonds. The resulting bicyclic peptide has greatly enhanced proteolytic stability as well as cell-permeability. Once inside the cell, the disulfide bonds are reduced to produce a linear, biologically active peptide. This strategy was applied to generate a cell-permeable bicyclic peptidyl inhibitor against the NEMO-IKK interaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/348,706, filed May 9, 2019, which is a U.S. national stageapplication filed under 35 U.S.C. § 371 of PCT/US2017/060881, filed Nov.9, 2017, which claims priority to U.S. Application No. 62/419,781, filedon Nov. 9, 2016, U.S. Application No. 62/425,550, filed Nov. 22, 2016,and U.S. Application No. 62/438,141, filed Dec. 22, 2016, each of whichare herein incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under GM110208,GM122459, and GM062820 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Compared to small-molecule drugs, peptides are highly selective andefficacious and, at the same time, relatively safe and well tolerated. Aparticularly exciting application of peptides is the inhibition ofprotein-protein interactions (PPIs), which remain challenging targetsfor small molecules. Consequently, there is an increased interest inpeptides in pharmaceutical research and development, and ˜140 peptidetherapeutics are currently being evaluated in clinical trials. However,peptides are inherently susceptible to proteolytic degradation.Additionally, peptides are generally impermeable to the cell membrane,largely limiting their applications to extracellular targets. AlthoughN-methylation of the peptide backbone and formation of intramolecularhydrogen bonds have been shown to improve the proteolytic stability andmembrane permeability of certain cyclic peptides (T. Rezai, et al., J.Am. Chem. Soc. 2006, 128, 14073), alternative strategies to increaseboth the metabolic stability and cell permeability of peptide drugs areclearly needed.

NF-κB is a transcription factor that controls the expression of numerousgene products involved in immune, stress, inflammatory responses, cellproliferation, and apoptosis (A. Oeckinghaus, S. Ghosh, Cold SpringHarb. Perspect. Biol. 2009, 1, a000034). Aberrant activation of NF-κBsignaling has been implicated in a number of autoimmune diseases (e.g.,rheumatoid arthritis) and cancer (e.g., diffuse large B-cell lymphoma),among others (V. Baud, M. Karin, Nat. Rev. Drug Discov. 2009, 8, 33;S.-C. Sun, et al., Trends Immunol. 2013, 34, 282; F. D. Herrington, etal., J. Biomol. Screen. 2016, 21, 223; G. Cildir, et al., Trends Mol.Med. 2016, 22, 414). Canonical NF-κB signaling is mediated by theinteraction between the inhibitor of κB (IκB)-kinase (IKK) complex andregulatory protein NF-κB essential modifier (NEMO) (S. Yamaoka, et al.,Cell 1998, 26, 1231; D. M. Rothwarf, et al., Nature 1998, 395, 297).Binding to NEMO activates IKK, which in turn phosphorylates IκB,promoting the proteasomal degradation of IκB and release of activeNF-κB. Modulators targeting various steps of the NF-κB signaling pathwayhave been reported, and some of them have progressed into the clinic (V.Baud, M. Karin, Nat. Rev. Drug Discov. 2009, 8, 33; S.-C. Sun, et al.,Trends Immunol. 2013, 34, 282; F. D. Herrington, et al., J. Biomol.Screen. 2016, 21, 223; G. Cildir, et al., Trends Mol. Med. 2016, 22,414; S. C. Gupta, et al., Biochim. Biophys. Acta. 2011, 1799, 775; T. M.Herndon, et al., Clin. Cancer Res. 2013, 19, 4559). One attractivestrategy for ameliorating the NF-κB activity is to selectively disruptthe IKK-NEMO interaction. Previous studies generated a weak NEMOinhibitor (K_(D)˜37 μM), Antp-NBD (Table 6, peptide 1), which containsthe 11-residue NEMO-binding domain (NBD) of IKKβ covalently linked to acell-penetrating peptide (CPP), Antp (M. J. May, et al., Science 2000,289, 1550). Interestingly, Antp-NBD blocks the IKK activity stimulatedby different pro-inflammatory stimuli, but does not affect the basalNF-κB activity, thus providing a potentially safe and effectivemechanism for reducing aberrant NF-κB activity (J. May, et al., Science2000, 289, 1550). In several pre-clinical studies, Antp-NBD demonstratedin vivo efficacy for treating Duchenne muscular dystrophy and largeB-cell lymphoma in mouse and canine models (E. Jimi, et al., Nat. Med.2004, 10, 617; S. Dai, et al., J. Biol. Chem. 2004, 279, 37219; W.Shibata, et al., J. Immunol. 2007, 179, 2681; S. H. Dave, et al., J.Immunol. 2007, 179, 7852; A. Gaurnier-Hausser, et al., Clin. Cancer Res.2011, 17, 4661; J. M. Peterson, et al., Mol. Med. 2011, 17, 508; D. A.Delfin, et al., J. Transl. Med. 2011, 9, 68; D. P. Reay, et al.,Neurobiol. Dis. 2011, 43, 598; J. N. Kornegay, et al., Skelet. Muscle2014, 4, 18; G. Habineza Ndikuyeze, et al., PLoS One, 2014, 9, e95404).However, to achieve clinical utility, Antp-NBD would benefitsignificantly from improvements in its NEMO-binding affinity, metabolicstability, and cell-permeability. What are thus needed are newcompositions and methods for modulating NF-κB signaling. Thecompositions and methods disclosed herein address these and other needs.

SUMMARY

Disclosed herein are compounds, compositions, methods for making andusing such compounds and compositions. In various embodiments disclosedherein are bicyclic peptides, compositions comprising such bicyclicpeptides, and methods of making and using them.

In some embodiments, the bicyclic peptides disclosed herein comprise:(a) a first cyclic peptide comprising a cell-penetrating peptidesequence (X_(m)); (b) a second cyclic peptide comprising a peptidylligand (X_(n)); and (c) at least one disulfide bond which forms at leastone of the first cyclic peptide or the second cyclic peptide, whereinthe first cyclic peptide is conjugated to the second cyclic peptide.

In various embodiments, X_(m) comprises the following peptide sequence:-AA¹-AA²-AA³-AA⁴-AA⁵-(AA⁶)_(m)-(AA⁷)_(n)-(AA⁸)_(p)-(AA⁹)_(q)-

wherein:

-   -   AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹, are each        independently an amino acid, which is optionally substituted,        and where at least three amino acids are arginine and at least        two amino acids independently comprise a hydrophobic side chain;        and    -   m, n, p, and q are independently selected from 0 and 1.

In some embodiments, the bicyclic peptides comprise a linking moietywhich is conjugated, directly or indirectly, to X_(m) and X_(n). In someembodiments, X_(m) is cyclized through the linking moiety, the C- orN-terminus of X_(n) is conjugated to the linking moiety, and X_(n) iscyclized through the disulfide bond, thereby forming the bicyclicpeptide. In other embodiments, the linking moiety is conjugated to theside chain of an amino acid in the first cyclic peptide, the C- orN-terminus of X_(n) is conjugated to the linking moiety, and whereinX_(n) is cyclized through the disulfide bond, thereby forming thebicyclic peptide.

In some embodiments, the bicyclic peptides disclosed herein comprise afirst disulfide bond and a second disulfide bond. In other embodiments,linking moiety comprises a first substituent which forms the firstdisulfide bond and a second substituent which forms the second disulfidebond. In some embodiments, X_(m) is cyclized through the first disulfidebond, and X_(n) is cyclized through the second disulfide bond, therebyforming the bicyclic peptide. In still other embodiments, the linkingmoiety is conjugated to the side chain of the first cyclic peptide, andX_(n) is cyclized through the first disulfide bond and the seconddisulfide bond, thereby forming the bicyclic peptide. In yet still otherembodiments, the C- or N-terminus of X_(m) is conjugated to the linkingmoiety and X_(m) is cyclized through the first disulfide bond, andwherein the C- or N-terminus of X_(n) is conjugated to the linkingmoiety and X_(n) is cyclized through the second disulfide bond, therebyforming the bicyclic peptide. In still even more embodiments, the C- orN-terminus of X_(n) is conjugated to the linking moiety, and whereinX_(m) is cyclized through the first disulfide bond and X_(n) is cyclizedthrough the second disulfide bond, thereby forming the bicyclic peptide.

In some embodiments, the bicyclic peptides disclosed herein comprise athird disulfide bond. In other embodiments, the linker moiety comprisesa first substituent which forms the first disulfide bond, a secondsubstituent which forms the second disulfide bond, and third substituentwhich forms the third disulfide bond. In still other embodiments, X_(m)is fused to X_(n), thereby forming a fused X_(m)—X_(n) peptide, and thelinking moiety is conjugated to the fused X_(m)—X_(n) preptide throughthe third disulfide bond, and wherein X_(m) is cyclized through thefirst disulfide bond and X_(n) is cyclized through the second disulfidebond, thereby forming the bicyclic peptide.

In some embodiments, the bicyclic peptides disclosed herein comprise afourth disulfide bond. In some embodiments, the linker moiety comprisesa first substituent which forms the first disulfide bond, a secondsubstituent which forms the second disulfide bond, third substituentwhich forms the third disulfide bond comprises, and a fourth substituentwhich forms a forth disulfide bond. In other embodiments, X_(m) iscyclized through the first disulfide bond and the second disulfide bond,and X_(n) is cyclized through the third disulfide bond and the fourthdisulfide bond, thereby forming the bicyclic peptide.

In various embodiments, the bicyclic peptides disclosed herein have astructure according to any of Formulae 1-12

Number Formula 1

2

3

4

5

6

7

8

9

10

11

12

wherein:

-   -   AA^(S) at each occurrence is independently a moiety which forms        a disulfide bond with J;    -   L-J is the linking moeity, wherein:        -   J is absent, or an alkyl, N-alkyl, alkenyl, alkynyl,            carbocyclyl, or heterocyclyl, each of which are            independently substituted with at least two substituents            which independently form a disulfide bond with AA^(S) at            each occurrence; and        -   L is absent or a moiety which links AA^(S) to an amino acid            in X_(m), X_(n), or a combination thereof; and

SS at each instance represents a disulfide bond.

In some embodiments, the bicyclic peptides described above have astructure according to any of Formula I, II, V, VI, VII, VIII, IX, X,and XII, each of which are described in more detail below.

In embodiments, disclosed herein are bicyclic peptides comprisingFormula I or II

wherein X_(m) and X_(n) independently comprise a sequence of 1-20 aminoacids and R¹ is OH, OR², or NHR², wherein R² is a C₁₋₂₀ alkyl, C₆₋₁₀aryl or heteroaryl, amino acid, peptide sequence of 2 to 20 amino acids,detectable moiety, or solid support.

As used herein, X_(m) refers to a cell penetrating peptide sequence. Insome embodiments, X_(m) is from 5 to 10 amino acids in length. Infurther embodiments, at least one, at least two, or at least three aminoacids in X_(m) have a hydrophobic side chain. In certain embodiments,X_(m) comprises one or more phenylalanine, naphthylalanine, tryptophan,or an analog or derivative thereof. In some embodiments, X_(m) comprisesat least one arginine or an analog or derivative thereof. In otherembodiments, X_(m) comprises a sequence listed in Table 2 (SEQ ID NO:62through SEQ ID NO:146). In certain embodiments, X_(m) is or comprisesRRRRΦF or FΦRRRR.

As used herein, X_(n) refers to a cargo sequence. In some embodiments,X_(n) comprises a sequence listed in Table 5 (SEQ ID NO:147 through SEQID NO:159). In some embodiments, the bicyclic peptide has a sequencelisted in Table 6 (SEQ ID NO:160 through SEQ ID NO:167).

In additional examples disclosed herein are peptides of Formula IIIBMB-(AA^(n))_(n)   IIIwherein n is an integer of from 5 to 20, and each AA^(n) is,independently, a natural or non-natural amino acid residue, with atleast two AA^(n) residues being cysteine, and BMB is a3,5-bis(mercaptomethyl)benzoic acid residue. In some specific examples uis 4 to 20, 5 to 9, 6 to 9, 7 to 8, or 8 to 9.

Also disclosed herein, in various embodiments, are methods of making abicyclic peptide, comprising:

(a) contacting a solid supported peptide having from 8 to 40 amino acid,wherein at least two amino acids are independently selected from thegroup consisting of a cysteine, homocysteine, an amino acid analoghaving a thiol group, with a compound of Formula IV:

-   -   wherein Q¹ and Q² are, independent of one another, chosen from        CH or N; and        (b) cleaving the peptide from the solid support.

Other embodiments of the present disclosure provide a bicyclic peptidecomprising Formula V, VI, VII, VIII, IX, X, or XII:

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹ are each        independently an amino acid, which is optionally substituted,        where at least three amino acids are arginine and at least two        amino acids independently comprise a hydrophobic side chain;    -   m, n, p, and q are independently selected from 0 and 1;    -   AA¹⁰ and AA¹¹, are each independently an amino acid, which is        optionally substituted;    -   b and c are independently an integer from 0 to 20;    -   AA^(S) at each occurrence is independently a moiety which forms        a disulfide bond with J;    -   J is an alkyl, N-alkyl, alkenyl, alkynyl, carbocyclyl, or        heterocyclyl, each of which are independently substituted with        at least two substituents which independently form a disulfide        bond with AA^(S) at each occurrence;    -   ss at each instance represents a disulfide bond; and    -   L is a moiety which links J to an amino acid, X_(n), or a        combination thereof; and    -   X_(n) is a cargo moiety comprising a peptide sequence having        from 1 to 20 amino acids.

In some embodiments, J is N-alkyl, aryl, or heteroaryl, each of whichare independently substituted with at least two substituents whichindependently form a disulfide bond with AA^(S) at each occurrence. Inother embodiments, J is

In some embodiments, L is a bond, an amino acid,

wherein a is an integer from 0 to 10.

In some embodiments, each AA^(S) independently is

wherein the C-terminus of AA^(S) forms an amide bond or is R¹, whereinR¹ is OH, OR², NHR²;and wherein R² is an alkyl, aryl, heteroaryl, amino acid, peptidesequence of 2 to 20 amino acids, detectable moiety, or solid support.

In some embodiments, the bicyclic peptides disclosed herein are selectedfrom the group consisting of:

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein each d is independently 1 or 2; and    -   wherein R¹ is OH, OR², NHR²; and R² is a alkyl, aryl,        heteroaryl, amino acid residue, peptide sequence of 2 to 20        amino acid residues, detectable moiety, or solid support.

In some embodiments, the at least two amino acid which independentlycomprise a hydrophobic side chain are selected from the group consistingof glycine, phenylglycine, alanine, valine, leucine, isoleucine,norleucine, phenylalanine, tryptophan, naphthylalanine, proline, andcombinations thereof, wherein the aromatic side chains on phenylglycine,phenylalanine, tryptophan, or naphthylalanine are each optionallysubstituted with a halogen. In other embodiments, the at least two aminoacid which independently comprise a hydrophobic side chain areindependently selected from the group consisting of phenylalanine,naphthylalanine, and combinations thereof. In still other embodiments,the at least two amino acids which independently comprise a hydrophobicresidue are consecutive amino acids.

In some embodiments:

-   -   AA¹ is L arginine;    -   AA² is L-arginine;    -   AA³ is L-arginine;    -   AA⁴ is L-phenylalanine;    -   AA⁵ is L phenylalanine; and    -   m, n, p, and q, are each 0.

or

-   -   AA¹ is L-phenylalanine;    -   AA² is L-naphthylalanine;    -   AA³ is L-arginine;    -   AA⁴ is L-arginine;    -   AA⁵ is L-arginine;    -   m is 1 and AA⁶ is L-arginine; and    -   n, p, and q are each 0.

or

-   -   AA¹ is L-arginine;    -   AA² is L-arginine;    -   AA³ is L-arginine;    -   AA⁴ is L-arginine;    -   AA⁵ is L-naphthylalanine;    -   m is 1 and AA⁶ is L-phenylalanine; and    -   n, p, and q are each 0.

In some embodiments, at least three consecutive amino acids havealternating chirality. In other embodiments, the at least threeconsecutive amino acids having alternating chirality are arginines.

In some embodiments:

-   -   AA¹ is D-phenylalanine;    -   AA² is L-naphthylalanine;    -   AA³ is L-arginine;    -   AA⁴ is D-arginine;    -   AA⁵ is L-arginine;    -   m is 1 and AA⁶ is D-arginine; and    -   n, p, and q are each 0.

or

-   -   AA¹ is D-phenylalanine;    -   AA² is L-naphthylalanine;    -   AA³ is L-arginine;    -   AA⁴ is D-arginine;    -   AA⁵ is L-arginine;    -   m and n are each 1, and AA⁶ is D-arginine and AA⁷ is L-arginine;        and    -   p and q are each 0.

or

-   -   AA¹ is D-phenylalanine;    -   AA² is L-naphthylalanine;    -   AA³ is L-arginine;    -   AA⁴ is D-arginine;    -   AA⁵ is L-arginine;    -   m and n are each 1, and AA⁶ is D-arginine and AA⁷ is        L-phenylalanine; and    -   p and q are each 0.

In other embodiments, AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹ isselected from SEQ ID NO:62 through SEQ ID NO:146.

In some embodiments, the peptide sequence in X_(n) inhibits at least oneprotein-protein interaction. In other embodiments, the protein-proteininteraction is an interaction between a κB-kinase (IKK) complex and aregulatory protein NF-κB essential modifier (NEMO). In still otherembodiments, the peptide sequence in X_(n) is an inhibitor against Ras,PTP1 B, Pin 1, Grb2 SH2, MDM2, or combinations thereof. In yet stillother embodiments, the peptide sequence in X_(n) is a wild-type peptidylligand or a peptide mimetic.

In some embodiments, the provided herein are compounds according toFormula V-A, VI-A, VII-A, VIII-A, IX-A, X-A, or XII-A and XII-B:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, AA⁹, AA¹⁰, and AA¹¹ are        independently selected from an amino acid, which is optionally        substituted, where are at least three amino acids are arginine,        and at least two amino acids independently comprise a        hydrophobic side chain;    -   m, n, p, or q are independently selected from 0 and 1;    -   b and c are independently an integer from 0 to 20;    -   AA^(S)′ at each occurrence is independently a moiety which        comprises a thiol;    -   J′ is an alkyl, N-alkyl, alkenyl, alkynyl, carbocyclyl, or        heterocyclyl, each of which are independently substituted with        at least two thiol substituents; and    -   L is an optional moiety which links AA^(S)′ to an amino acid,        X_(n), or a combination thereof; and    -   X_(n) is a cargo moiety comprising a peptide sequence having        from 4 to 20 amino acids.

In some embodiments, J′ is N-alkyl, aryl, or heteroaryl. In furtherembodiments, J′ is

In some embodiments, L is absent, an amino acid,

wherein a is an integer from 0 to 20.

In some embodiments, each AA^(S)′ is independently:

-   -   wherein the C-terminus of AA^(S)′ forms an amide bond or is R¹,        wherein R¹ is OH, OR², NHR²; and wherein R² is a alkyl, aryl,        heteroaryl, amino acid residue, peptide sequence of 2 to 20        amino acid residues, detectable moiety, or solid support.

In some embodiments, the compounds have a structure selected from thegroup consisting of

or a pharmaceutically acceptable salt thereof, wherein each d isindependently 1 or 0.

In embodiments of the above compounds, the at least two amino acidswhich independently comprise a hydrophobic side chain are selected fromthe group consisting of glycine, phenylglycine, alanine, valine,leucine, isoleucine, norleucine, phenylalanine, tryptophan,naphthylalanine, proline, and combinations thereof, wherein the aromaticresidues on phenylglycine, phenylalanine, tryptophan, naphthylalanineare optionally substituted. In other embodiments, the at least two aminoacids which independently comprise a hydrophobic side chain are selectedfrom the group consisting of phenylalanine, naphthylalanine, andcombinations thereof. In still other embodiments, the at least two aminoacids which independently comprise a hydrophobic side chain areconsecutive amino acids.

In some embodiments:

-   -   AA¹ is L arginine;    -   AA² is L-arginine;    -   AA³ is L-arginine;    -   AA⁴ is L-phenylalanine;    -   AA⁵ is L phenylalanine; and    -   m, n, p, and q, are each 0.

or

-   -   AA¹ is L-phenylalanine;    -   AA² is L-naphthylalanine;    -   AA³ is L-arginine;    -   AA⁴ is L-arginine;    -   AA⁵ is L-arginine;    -   m is 1 and AA⁶ is L-arginine; and    -   n, p, and q are each 0.

or

-   -   AA¹ is L-arginine;    -   AA² is L-arginine;    -   AA³ is L-arginine;    -   AA⁴ is L-arginine;    -   AA⁵ is L-naphthylalanine;    -   m is 1 and AA⁶ is L-phenylalanine; and    -   n, p, and q are each 0.

In some embodiments, at least three consecutive amino acids havealternating chirality. In other embodiments, the at least threeconsecutive amino acids having alternating chirality are arginines.

In some embodiments:

-   -   AA¹ is D-phenylalanine;    -   AA² is L-naphthylalanine;    -   AA³ is L-arginine;    -   AA⁴ is D-arginine;    -   AA⁵ is L-arginine;    -   m is 1 and AA⁶ is D-arginine; and    -   n, p, and q are each 0.

or

-   -   AA¹ is D-phenylalanine;    -   AA² is L-naphthylalanine;    -   AA³ is L-arginine;    -   AA⁴ is D-arginine;    -   AA⁵ is L-arginine;    -   m and n are each 1, and AA⁶ is D-arginine and AA⁷ is L-arginine;        and    -   p and q are each 0.

or

-   -   AA¹ is D-phenylalanine;    -   AA² is L-naphthylalanine;    -   AA³ is L-arginine;    -   AA⁴ is D-arginine;    -   AA⁵ is L-arginine;    -   m and n are each 1, and AA⁶ is D-arginine and AA⁷ is        L-phenylalanine; and    -   p and q are each 0.

In other embodiments, AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹ isselected from SEQ ID NO:62 through SEQ ID NO:146.

In some embodiments, the peptide sequence in X_(n) inhibits at least oneprotein-protein interaction. In further embodiments, the protein-proteininteraction is an interaction between a κB-kinase (IKK) complex and aregulatory protein NF-κB essential modifier (NEMO). In otherembodiments, the peptide sequence in X_(n) is an inhibitor against Ras,PTP1 B, Pin 1, Grb2 SH2, MDM2, or combinations thereof. In still otherembodiments, the peptide sequence in X_(n) is a peptidyl is a wild-typepeptide ligand or a peptide mimetic.

In various embodiments, a compound according to Formula XI is disclosed:

wherein:

-   -   Y at each instance is independently CH, N, O or S, provided no        more than four Y are N, O, S, or combinations thereof;    -   Z is OR_(a), hydrogen, halogen, carbocyclyl, heterocyclyl, or an        amino acid;    -   R at each instance is independently an alkyl, alkenyl, alkynyl,        carbocyclyl, heterocyclyl, or an amino acid; and    -   R_(a) is independently H, C(O)alkyl, alkyl, alkenyl, alkynyl,        carbocyclic, or heterocyclyl.

In some embodiments, Y is independently CH. In other embodiments, thecompound has a structure according to Formula XI-A:

In some embodiments, each R is independently aryl or hetereoaryl. Infurther embodiments, the compound has a structure according to FormulaXI-B:

wherein Q at each instance is independently CH or N.

In some embodiments, Z is OH. In other embodiments, the compound has thefollowing structure:

Also disclosed herein are methods delivering a therapeutic agent tocytoplasm of a cell, comprising administering a compound of any one ofFormulas V-XII.

Also disclosed are pharmaceutical compositions comprising a bicyclicpeptide disclosed herein and a pharmaceutical carrier

Also disclosed herein are methods of treating or preventing a disorderin a subject, such as a human, comprising administering to the subjectan effective amount of a compound disclosed herein or a pharmaceuticallyacceptable salt thereof. In some examples, the subject is an animal,such as a human. In some examples, the subject is identified as having aneed for treatment of the disorder. In some examples, the method treatsa disorder. In some examples, the disorder is associated with aberantNF-κB signaling. In some examples the disorder is associated withuncontrolled cellular proliferation, such as cancer. In some examples,the disorder is cancer. In some examples the disorder is an inflammatorydisorder, such as irritable bowl syndrome. In some examples, thedisorder is an autoimmune disorder, such as a disorder selected fromrheumatoid arthritis, ankylosing spondylitis, Crohn's disease,psoriasis, hidradenitis suppurativa, and refractory asthma. In somefurther examples, disclosed herein is a method of treating Duchennemuscular dystrophy or large B-cell lymphoma.

Also disclosed herein is a method for identifying a drug candidate fortreatment of a disorder, the method comprising the steps of: exposing acompound disclosed herein, a compound prepared by the methods disclosedherein, a library disclosed herein, or a library prepared by the methodsdisclosed to a receptor associated with the disorder; b) detectingreaction between the receptor and the compound or the library; and c)determining the identity of compound reacting with the receptor.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

DESCRIPTION OF FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1A shows MFI of HeLa cells after 2-h treatment with 5 μMFITC-labeled peptide cFΦR4 (SEQ ID NO.:68) or 1-5, as determined by flowcytometry analysis. Blank, no peptide. FIG. 1B shows inhibition of theNEMO-IKKγ interaction by peptides 1, 4, and 5 as monitored by the HTRFassay. FIG. 1C shows dose-dependent inhibition of TNFα inducedactivation of NF-κB signaling in HEK293 cells by peptides 1, 4, and 5.FIG. 1D shows a comparison of the serum stability of peptides 1, 4, and6. Data reported are the mean±SD of three independent experiments.

FIG. 2 is a reversible peptide bicyclization strategy. GSH, glutathione.

FIG. 3 shows the structures of FITC-labeled peptides 2 and 3.

FIG. 4 shows the structures of peptides 4, 5, and 6.

FIG. 5A is a preparative reversed-phase HPLC chromatogram showing thepurification of crude peptide 4 following solid-phase synthesis andtrituration with diethyl ether. FIG. 5B is an analytical reversed-phaseHPLC and MALDI-TOF MS analysis of purified peptide 4 before and afterincubation with 5 mM glutathione in phosphate buffer (pH 7.4) at RT for2 h.

FIG. 6 shows the inhibition of the NEMO-IKKγ interaction by peptidesRRRRΦFCALDWSWLQC (IC₅₀=1.4 μM) (SEQ ID NO.:215) and RRRRΦFTALDWSWLQT(IC₅₀=1.6 μM) (SEQ ID NO.:216) as monitored by the HTRF assay.

FIG. 7 is an analytical reversed-phase HPLC showing the purity of otherpeptides used in this work. The authenticity of the peptides wasconfirmed by MALDI-TOF MS analysis.

FIG. 8 is a schematic illustration of a tris-(disulfide) containingbicyclic peptide.

FIG. 9 is a schematic illustration of a bis-(disulfide) containingbicyclic peptide which releases the cargo from the cycliccell-penetrating peptide upon entry into the cytosole of a cell.

FIG. 10A shows the structure of a bicyclic peptide comprising cycliccomprising CPP12 (FfΦRrRr) conjugated to a peptidyl inhibitor againstKeap1-Nrf2, having a fluorescent label (NFL). FIG. 10B shows a structureof the linear peptidyl inhibitor against Keap1-Nrf2, having afluorescent label (NFL) in the absence of a cyclic CPP.

FIG. 11 shows the structure of a bicyclic peptide comprising cycliccomprising a cyclic CPP12 (FfΦRrRr) conjugated to a peptidyl inhibitoragainst Pin1, having a fluorescent label (NFL).

FIG. 12A shows the structure of a bicyclic peptide comprising cycliccomprising a cyclic CPP12 (FfΦRrRr) conjugated to a peptidyl inhibitoragainst the CAL PDZ-CFTR interaction, having a fluorescent label (NFL).FIG. 12B shows a structure of the linear peptidyl inhibitor against theCAL PDZ-CFTR interaction, having a fluorescent label (NFL) in theabsence of a cyclic CPP.

FIG. 13A shows the structure of a bicyclic peptide comprising cycliccomprising a cyclic CPP12 (FfΦRrRr) conjugated to a peptidyl inhibitoragainst the MDM2-p53 interaction (PMI), having a fluorescent label(NFL). FIG. 13B shows a structure of the linear peptidyl inhibitoragainst the PMI, having a fluorescent label (NFL) in the absence of acyclic CPP.

FIG. 14 graphically illustrates the cytosolic uptake efficiency ofunconjugated peptidyl inhibitors compared to the cyclic CPP-peptideconjugates (i.e., the bicyclic peptides of the present disclosure).

FIG. 15A shows the structure of a bicyclic peptide comprising cycliccomprising a cyclic CPP12 (FfΦRrRr) conjugated to a peptidyl inhibitoragainst Keap1-Nrf2. FIG. 15B shows a structure of the linear peptidylinhibitor against Keap1-Nrf2 in the absence of a cyclic CPP.

FIG. 16 shows the structure of a bicyclic peptide comprising cycliccomprising a cyclic CPP12 (FfΦRrRr) conjugated to a peptidyl inhibitoragainst Pin1.

FIG. 17 graphically illustrates the serum stability of bicyclic peptidecomprising a cyclic CPP12 (FfΦRrRr) conjugated to a peptidyl inhibitoragainst the MDM2-p53 interaction (PMI), compared to that of the linearpeptidyl inhibitor against the PMI.

FIG. 18 graphically illustrates the serum stability of a bicyclicpeptide comprising cyclic CPP12 (FfΦRrRr) conjugated to a peptidylinhibitor against the Keap1-Nrf2 interaction, compared to that of thelinear peptidyl inhibitor against the Keap1-Nrf2.

FIG. 19 graphically illustrates the serum stability of a bicyclicpeptide comprising CPP12 (FfΦRrRr) conjugated to a peptidyl inhibitoragainst Pin-1 (P1), compared to that of linear peptidyl inhibitoragainst P1.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examples andFigures included therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

General Definitions

As used herein, nomenclature for compounds, including organic compounds,can be given using common names, IUPAC, IUBMB, or CAS recommendationsfor nomenclature. When one or more stereochemical features are present,Cahn-Ingold-Prelog rules for stereochemistry can be employed todesignate stereochemical priority, E/Z specification, and the like. Oneof skill in the art can readily ascertain the structure of a compound ifgiven a name, either by systemic reduction of the compound structureusing naming conventions, or by commercially available software, such asCHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “subject” refers to the target ofadministration, e.g. a subject. Thus the subject of the herein disclosedmethods can be a vertebrate, such as a mammal, a fish, a bird, areptile, or an amphibian. Alternatively, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig, fish, bird, or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In some examples, the subject is a mammal. A patient refers toa subject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects. In some examples of thedisclosed methods, the subject has been diagnosed with a need fortreatment of cancer prior to the administering step. In some examples ofthe disclosed method, the subject has been diagnosed with cancer priorto the administering step. The term subject also includes a cell, suchas an animal, for example human, cell.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. In some examples, the term covers any treatmentof a subject, including a mammal (e.g., a human), and includes: (i)preventing the disease from occurring in a subject that can bepredisposed to the disease but has not yet been diagnosed as having it;(ii) inhibiting the disease, i.e., arresting its development; or (iii)relieving the disease, i.e., causing regression of the disease. In someexamples, the subject is a mammal such as a primate, and, in someexamples, the subject is a human. The term “subject” also includesdomesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle,horses, pigs, sheep, goats, fish, bird, etc.), and laboratory animals(e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. For example,“diagnosed with cancer” means having been subjected to a physicalexamination by a person of skill, for example, a physician, and found tohave a condition that can be diagnosed or treated by a compound orcomposition that can treat or prevent cancer. As a further example,“diagnosed with a need for treating or preventing cancer” refers tohaving been subjected to a physical examination by a person of skill,for example, a physician, and found to have a condition characterized bycancer or other disease wherein treating or preventing cancer would bebeneficial to the subject.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder (e.g., a disorder relatedto cancer) based upon an earlier diagnosis by a person of skill andthereafter subjected to treatment for the disorder. It is contemplatedthat the identification can, In some examples, be performed by a persondifferent from the person making the diagnosis. It is also contemplated,in some examples, that the administration can be performed by one whosubsequently performed the administration.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, sublingual administration, buccal administration, andparenteral administration, including injectable such as intravenousadministration, intra-arterial administration, intramuscularadministration, and subcutaneous administration. Administration can becontinuous or intermittent. In some examples, a preparation can beadministered therapeutically; that is, administered to treat an existingdisease or condition. In some examples, a preparation can beadministered prophylactically; that is, administered for prevention of adisease or condition.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, target receptor, or other biological entitytogether in such a manner that the compound can affect the activity ofthe target (e.g., receptor, transcription factor, cell, etc.), eitherdirectly; i.e., by interacting with the target itself, or indirectly;i.e., by interacting with another molecule, co-factor, factor, orprotein on which the activity of the target is dependent.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeadverse side effects. The specific therapeutically effective dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of a compound at levels lower than those required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved. If desired, the effective dailydose can be divided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts orsubmultiples thereof to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. In some examples, a preparation can beadministered in a “prophylactically effective amount”; that is, anamount effective for prevention of a disease or condition.

As used herein, “EC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a compound or a drug) that is required for50% enhancement or activation of a biological process, or component of aprocess, including a protein, subunit, organelle, ribonucleoprotein,etc. EC₅₀ also refers to the concentration or dose of a substance thatis required for 50% enhancement or activation in vivo, as furtherdefined elsewhere herein. Alternatively, EC₅₀ can refer to theconcentration or dose of compound that provokes a response halfwaybetween the baseline and maximum response. The response can be measuredin an in vitro or in vivo system as is convenient and appropriate forthe biological response of interest. For example, the response can bemeasured in vitro using cultured muscle cells or in an ex vivo organculture system with isolated muscle fibers. Alternatively, the responsecan be measured in vivo using an appropriate research model such asrodent, including mice and rats. The mouse or rat can be an inbredstrain with phenotypic characteristics of interest such as obesity ordiabetes. As appropriate, the response can be measured in a transgenicor knockout mouse or rat wherein the gene or genes has been introducedor knocked-out, as appropriate, to replicate a disease process.

As used herein, “IC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a compound or a drug) that is required for50% inhibition or diminuation of a biological process, or component of aprocess, including a protein, subunit, organelle, ribonucleoprotein,etc. IC₅₀ also refers to the concentration or dose of a substance thatis required for 50% inhibition or diminuation in vivo, as furtherdefined elsewhere herein. Alternatively, IC₅₀ also refers to the halfmaximal (50%) inhibitory concentration (IC) or inhibitory dose of asubstance. The response can be measured in an in vitro or in vivo systemas is convenient and appropriate for the biological response ofinterest. For example, the response can be measured in vitro usingcultured muscle cells or in an ex vivo organ culture system withisolated muscle fibers. Alternatively, the response can be measured invivo using an appropriate research model such as rodent, including miceand rats. The mouse or rat can be an inbred strain with phenotypiccharacteristics of interest such as obesity or diabetes. As appropriate,the response can be measured in a transgenic or knockout mouse or ratwherein a gene or genes has been introduced or knocked-out, asappropriate, to replicate a disease process.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompound disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds. Exemplary derivatives include salts, esters, amides, salts ofesters or amides, and N-oxides of a parent compound.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. Thus, an ethylene glycolresidue in a polyester refers to one or more —OCH₂CH₂O— units in thepolyester, regardless of whether ethylene glycol was used to prepare thepolyester. Similarly, a sebacic acid residue in a polyester refers toone or more —CO(CH₂)₈CO— moieties in the polyester, regardless ofwhether the residue is obtained by reacting sebacic acid or an esterthereof to obtain the polyester. As another example, an amino acidresidue, e.g., in a peptide, refers to one or more -AA- moeities, andsuch residues may be referred to herein interchangibly as an amino acidor an amino acid residue.

Chemical Definitions

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In some examples, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. It is also contemplated that, in certain examples,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Thealkyl group can be cyclic or acyclic. The alkyl group can be branched orunbranched. The alkyl group can also be substituted or unsubstituted.For example, the alkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether,halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.A “lower alkyl” group is an alkyl group containing from one to six(e.g., from one to four) carbon atoms.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” or “haloalkyl” specifically refers to analkyl group that is substituted with one or more halide, e.g., fluorine,chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refersto an alkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, alkyl, cycloalkyl, alkoxy,amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol asdescribed herein.

The term “polyalkylene group” as used herein is a group having two ormore CH₂ groups linked to one another. The polyalkylene group can berepresented by the formula —(CH₂)_(a)—, where “a” is an integer of from2 to 500.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl orcycloalkyl group bonded through an ether linkage; that is, an “alkoxy”group can be defined as —OA¹ where A¹ is alkyl or cycloalkyl as definedabove. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as —OA¹-OA² or—OA¹-(OA²)_(a)-OA³, where “a” is an integer of from 1 to 200 and A¹, A²,and A³ are alkyl and/or cycloalkyl groups.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A³A⁴)are intended to include both the E and Z isomers. This can be presumedin structural formulae herein wherein an asymmetric alkene is present,or it can be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, orthiol, as described herein.

“Carbocyclyl,” “carbocyclic ring” or “carbocycle” refers to a ringstructure, wherein the atoms which form the ring are each carbon.Carbocyclic rings can comprise from 3 to 20 carbon atoms in the ring.Carbocyclic rings include aryls and cycloalkyl, cycloalkenyl andcycloalkynyl as defined herein. The carbocyclic group can be substitutedor unsubstituted. The carbocyclic group can be substituted with one ormore groups including, but not limited to, alkyl, cycloalkyl, alkoxy,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onecarbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groupsinclude, but are not limited to, cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl,norbornenyl, and the like. The term “heterocycloalkenyl” is a type ofcycloalkenyl group as defined above, and is included within the meaningof the term “cycloalkenyl,” where at least one of the carbon atoms ofthe ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group andheterocycloalkenyl group can be substituted or unsubstituted. Thecycloalkenyl group and heterocycloalkenyl group can be substituted withone or more groups including, but not limited to, alkyl, cycloalkyl,alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be unsubstituted orsubstituted with one or more groups including, but not limited to,alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, asdescribed herein.

The term “cycloalkynyl” as used herein is a non-aromatic carbon-basedring composed of at least seven carbon atoms and containing at least onecarbon-carbon triple bound. Examples of cycloalkynyl groups include, butare not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and thelike. The term “heterocycloalkynyl” is a type of cycloalkenyl group asdefined above, and is included within the meaning of the term“cycloalkynyl,” where at least one of the carbon atoms of the ring isreplaced with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkynyl group andheterocycloalkynyl group can be substituted or unsubstituted. Thecycloalkynyl group and heterocycloalkynyl group can be substituted withone or more groups including, but not limited to, alkyl, cycloalkyl,alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiolas described herein. The term “biaryl” is a specific type of aryl groupand is included in the definition of “aryl.” Biaryl refers to two arylgroups that are bound together via a fused ring structure, as innaphthalene, or are attached via one or more carbon-carbon bonds, as inbiphenyl.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” is a short hand notation for acarbonyl group, i.e., C═O.

The terms “amine” or “amino” as used herein are represented by theformula —NA¹A², where A¹ and A² can be, independently, hydrogen oralkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “alkylamino” as used herein is represented by the formula—NH(-alkyl) where alkyl is a described herein. Representative examplesinclude, but are not limited to, methylamino group, ethylamino group,propylamino group, isopropylamino group, butylamino group, isobutylaminogroup, (sec-butyl)amino group, (tert-butyl)amino group, pentylaminogroup, isopentylamino group, (tert-pentyl)amino group, hexylamino group,and the like.

The term “dialkylamino” as used herein is represented by the formula—N(-alkyl)₂ where alkyl is a described herein. Representative examplesinclude, but are not limited to, dimethylamino group, diethylaminogroup, dipropylamino group, diisopropylamino group, dibutylamino group,diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)aminogroup, dipentylamino group, diisopentylamino group, di(tert-pentyl)aminogroup, dihexylamino group, N-ethyl-N-methylamino group,N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH.

The term “ester” as used herein is represented by the formula —OC(O)A¹or —C(O)OA¹, where A¹ can be alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.The term “polyester” as used herein is represented by the formula-(A¹O(O)C-A²-C(O)O)_(a)— or -(A¹O(O)C-A²-OC(O))_(a)—, where A¹ and A²can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and“a” is an integer from 1 to 500. “Polyester” is as the term used todescribe a group that is produced by the reaction between a compoundhaving at least two carboxylic acid groups with a compound having atleast two hydroxyl groups.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group describedherein. The term “polyether” as used herein is represented by theformula -(A¹O-A²O)_(n)—, where A¹ and A² can be, independently, analkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group described herein and “a” is an integer of from 1 to500. Examples of polyether groups include polyethylene oxide,polypropylene oxide, and polybutylene oxide.

The term “halide” as used herein refers to the halogens fluorine,chlorine, bromine, and iodine.

The term “heterocycle,” as used herein refers to single and multi-cyclicaromatic or non-aromatic ring systems in which at least one of the ringmembers is other than carbon. Heterocycle includes azetidine, dioxane,furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole,including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole,piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran,tetrazine, including 1,2,4,5-tetrazine, tetrazole, including1,2,3,4-tetrazole and 1,2,4,5-tetrazole, thiadiazole, including,1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, thiazole,thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine,triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like.

“N-alkyl” refers to a alkyl radical as defined above containing at leastone nitrogen and where a point of attachment of the alkyl radical to therest of the molecule is through a nitrogen atom in the N-alkyl radical.Unless stated otherwise specifically in the specification, a N-alkylgroup can be optionally substituted.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group asdescribed herein.

The term “azide” as used herein is represented by the formula —N₃.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “nitrile” as used herein is represented by the formula —CN.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen or an alkyl,cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “sulfo-oxo” as used herein is represented by the formulas—S(O)A¹, —S(O)₂A¹, —OS(O)₂A¹, or —OS(O)₂OA¹, where A¹ can be hydrogen oran alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, or heteroaryl group as described herein. Throughout thisspecification “S(O)” is a short hand notation for S═O. The term“sulfonyl” is used herein to refer to the sulfo-oxo group represented bythe formula —S(O)₂A¹, where A¹ can be hydrogen or an alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl groupas described herein. The term “sulfone” as used herein is represented bythe formula A¹S(O)₂A², where A¹ and A² can be, independently, an alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein. The term “sulfoxide” as usedherein is represented by the formula A¹S(O)A², where A¹ and A² can be,independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “thiol” as used herein is represented by the formula —SH.

“R¹,” “R²,” “R³,” “R^(n),” where n is an integer, as used herein can,independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an alkyl group, a halide, and the like.Depending upon the groups that are selected, a first group can beincorporated within second group or, alternatively, the first group canbe pendant (i.e., attached) to the second group. For example, with thephrase “an alkyl group comprising an amino group,” the amino group canbe incorporated within the backbone of the alkyl group. Alternatively,the amino group can be attached to the backbone of the alkyl group. Thenature of the group(s) that is (are) selected will determine if thefirst group is embedded or attached to the second group.

As described herein, compounds can contain “optionally substituted”moieties. In general, the term “substituted,” whether preceded by theterm “optionally” or not, means that one or more hydrogens of thedesignated moiety are replaced with a suitable substituent. Unlessotherwise indicated, an “optionally substituted” group can have asuitable substituent at each substitutable position of the group, andwhen more than one position in any given structure can be substitutedwith more than one substituent selected from a specified group, thesubstituent can be either the same or different at every position.Combinations of substituents envisioned herein are preferably those thatresult in the formation of stable or chemically feasible compounds. Inis also contemplated that, in some examples, unless expressly indicatedto the contrary, individual substituents can be further optionallysubstituted (i.e., further substituted or unsubstituted).

The term “stable,” as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and, in some examples, their recovery,purification, and use for one or more of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO2;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘)) ₂; SiR^(∘) ₃; —(C₁₋₄ straight orbranched)alkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(∘)) ₂, wherein each R^(∘) may besubstituted as defined below and is independently hydrogen, C₁₋₆aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), ora 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(•), -(haloR^(•)),—(CH₂)₀₋₂OH, —(CH₂)O₂OR^(•), —(CH₂)₀₋₂CH(OR^(•))₂; —O(haloR^(•)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(•), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(•),—(CH₂)₀₋₂SR^(•), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(•),—(CH₂)₀₋₂NR^(•) ₂, —NO₂, —SiR^(•) ₃, —OSiR^(•) ₃, —C(O)SR, —(C₁₋₄straight or branched alkylene)C(O)OR, or —SSR^(•) wherein each R^(•) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN, —C(O)OH,—C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein each R^(•) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN,—C(O)OH, —C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein eachR^(•) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

The term “leaving group” refers to an atom (or a group of atoms) withelectron withdrawing ability that can be displaced as a stable species,taking with it the bonding electrons. Examples of suitable leavinggroups include halides and sulfonate esters, including, but not limitedto, triflate, mesylate, tosylate, and brosylate.

The terms “hydrolysable group” and “hydrolysable moiety” refer to afunctional group capable of undergoing hydrolysis, e.g., under basic oracidic conditions. Examples of hydrolysable residues include, withoutlimitation, acid halides, activated carboxylic acids, and variousprotecting groups known in the art (see, for example, “Protective Groupsin Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience,1999).

The term “organic residue” defines a carbon containing residue, i.e., aresidue comprising at least one carbon atom, and includes but is notlimited to the carbon-containing groups, residues, or radicals definedhereinabove. Organic residues can contain various heteroatoms, or bebonded to another molecule through a heteroatom, including oxygen,nitrogen, sulfur, phosphorus, or the like. Examples of organic residuesinclude but are not limited alkyl or substituted alkyls, alkoxy orsubstituted alkoxy, mono or di-substituted amino, amide groups, etc.Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15,carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbonatoms, or 1 to 4 carbon atoms. In some examples, an organic residue cancomprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbonatoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

A very close synonym of the term “residue” is the term “radical,” whichas used in the specification and concluding claims, refers to afragment, group, or substructure of a molecule described herein,regardless of how the molecule is prepared. For example, a2,4-thiazolidinedione radical in a particular compound has the structure

regardless of whether thiazolidinedione is used to prepare the compound.In some embodiments the radical (for example an alkyl) can be furthermodified (i.e., substituted alkyl) by having bonded thereto one or more“substituent radicals.” The number of atoms in a given radical is notcritical to the compounds and compositions disclosed herein unless it isindicated to the contrary elsewhere herein.

“Organic radicals,” as the term is defined and used herein, contain oneor more carbon atoms. An organic radical can have, for example, 1-26carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms,1-6 carbon atoms, or 1-4 carbon atoms. In some examples, an organicradical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbonatoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organicradicals often have hydrogen bound to at least some of the carbon atomsof the organic radical. One example, of an organic radical thatcomprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2-naphthylradical. In some embodiments, an organic radical can contain 1-10inorganic heteroatoms bound thereto or therein, including halogens,oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organicradicals include but are not limited to an alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, mono-substituted amino,di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy,alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl,thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl,substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclicradicals, wherein the terms are defined elsewhere herein. A fewnon-limiting examples of organic radicals that include heteroatomsinclude alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals,dimethylamino radicals and the like.

“Inorganic radicals,” as the term is defined and used herein, contain nocarbon atoms and therefore comprise only atoms other than carbon.Inorganic radicals comprise bonded combinations of atoms selected fromhydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, andhalogens such as fluorine, chlorine, bromine, and iodine, which can bepresent individually or bonded together in their chemically stablecombinations. Inorganic radicals have 10 or fewer, or preferably one tosix or one to four inorganic atoms as listed above bonded together.Examples of inorganic radicals include, but not limited to, amino,hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonlyknown inorganic radicals. The inorganic radicals do not have bondedtherein the metallic elements of the periodic table (such as the alkalimetals, alkaline earth metals, transition metals, lanthanide metals, oractinide metals), although such metal ions can sometimes serve as apharmaceutically acceptable cation for anionic inorganic radicals suchas a sulfate, phosphate, or like anionic inorganic radical. Inorganicradicals do not comprise metalloids elements such as boron, aluminum,gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gaselements, unless otherwise specifically indicated elsewhere herein.

As used herein, the symbol

(hereinafter can be referred to as “a point of attachment bond”) denotesa bond that is a point of attachment between two chemical entities, oneof which is depicted as being attached to the point of attachment bondand the other of which is not depicted as being attached to the point ofattachment bond. For example,

indicates that the chemical entity “XY” is bonded to another chemicalentity via the point of attachment bond. Furthermore, the specific pointof attachment to the non-depicted chemical entity can be specified byinference. For example, the compound CH₃—R³, wherein R³ is H or

infers that when R³ is “XY”, the point of attachment bond is the samebond as the bond by which R³ is depicted as being bonded to CH₃.

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the compounds andcompositions disclosed herein include all such possible isomers, as wellas mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture. Compounds describedherein can contain one or more asymmetric centers and, thus, potentiallygive rise to diastereomers and optical isomers. Unless stated to thecontrary, the compounds and compositions disclosed herein include allsuch possible diastereomers as well as their racemic mixtures, theirsubstantially pure resolved enantiomers, all possible geometric isomers,and pharmaceutically acceptable salts thereof Mixtures of stereoisomers,as well as isolated specific stereoisomers, are also included. Duringthe course of the synthetic procedures used to prepare such compounds,or in using racemization or epimerization procedures known to thoseskilled in the art, the products of such procedures can be a mixture ofstereoisomers.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and 1 or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture. Many of the compounds describedherein can have one or more chiral centers and therefore can exist indifferent enantiomeric forms. If desired, a chiral carbon can bedesignated with an asterisk (*). When bonds to the chiral carbon aredepicted as straight lines in the disclosed formulas, it is understoodthat both the (R) and (S) configurations of the chiral carbon, and henceboth enantiomers and mixtures thereof, are embraced within the formula.As is used in the art, when it is desired to specify the absoluteconfiguration about a chiral carbon, one of the bonds to the chiralcarbon can be depicted as a wedge (bonds to atoms above the plane) andthe other can be depicted as a series or wedge of short parallel linesis (bonds to atoms below the plane). The Cahn-Inglod-Prelog system canbe used to assign the (R) or (S) configuration to a chiral carbon.

Compounds described herein comprise atoms in both their natural isotopicabundance and in non-natural abundance. The disclosed compounds can beisotopically-labelled or isotopically-substituted compounds identical tothose described, but for the fact that one or more atoms are replaced byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature. Examples of isotopes thatcan be incorporated into compounds disclosed herein include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F and ³⁶Cl respectively.Compounds further comprise prodrugs thereof, and pharmaceuticallyacceptable salts of said compounds or of said prodrugs which contain theaforementioned isotopes and/or other isotopes of other atoms are withinthe scope of this invention. Certain isotopically-labeled compounds, forexample those into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., u isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labeled compounds and prodrugs thereof can generally beprepared by carrying out the procedures below, by substituting a readilyavailable isotopically labeled reagent for a non-isotopically labeledreagent.

The compounds described herein can be present as a solvate. In somecases, the solvent used to prepare the solvate is an aqueous solution,and the solvate is then often referred to as a hydrate. The compoundscan be present as a hydrate, which can be obtained, for example, bycrystallization from a solvent or from aqueous solution. In thisconnection, one, two, three or any arbitrary number of solvate or watermolecules can combine with the compounds disclosed herein to formsolvates and hydrates. Unless stated to the contrary, all such possiblesolvates are included in the discussion herein.

The term “co-crystal” means a physical association of two or moremolecules which owe their stability through non-covalent interaction.One or more components of this molecular complex provide a stableframework in the crystalline lattice. In certain instances, the guestmolecules are incorporated in the crystalline lattice as anhydrates orsolvates, see e.g. “Crystal Engineering of the Composition ofPharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a NewPath to Improved Medicines?” Almarasson, O., et. al., The Royal Societyof Chemistry, 1889-1896, 2004. Examples of co-crystals includep-toluenesulfonic acid and benzenesulfonic acid.

It is also appreciated that certain compounds described herein can bepresent as an equilibrium of tautomers. For example, ketones with anα-hydrogen can exist in an equilibrium of the keto form and the enolform.

Likewise, amides with an N-hydrogen can exist in an equilibrium of theamide form and the imidic acid form. Unless stated to the contrary, allsuch possible tautomers are included herein.

It is known that chemical substances form solids which are present indifferent states of order which are termed polymorphic forms ormodifications. The different modifications of a polymorphic substancecan differ greatly in their physical properties. The compounds can bepresent in different polymorphic forms, with it being possible forparticular modifications to be metastable. Unless stated to thecontrary, all such possible polymorphic forms are included.

In some examples, a structure of a compound can be represented by aformula:

which is understood to be equivalent to a formula:

wherein n is typically an integer. That is, R^(n) is understood torepresent five independent substituents, R^(n(a)), R^(n(b)), R^(n(c)),R^(n(d)), R^(n(e)). By “independent substituents,” it is meant that eachR substituent can be independently defined. For example, if in oneinstance R^(n(a)) is halogen, then R^(n(b)) is not necessarily halogenin that instance.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositionsdisclosed herein as well as the compositions themselves to be usedwithin the methods disclosed herein. These and other materials aredisclosed herein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions disclosed herein. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods disclosedherein.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

Abbreviations used herein are as follows: Cpa, L-4-chlorophenylalanine;dap, D-2,3-diaminopropionic acid; Dap, L-2,3-diaminopropionic acid;FITC, fluorescein isothiocyanate; Fpa, L-4-fluorophenylalanine; F₂pa,L-3,4-difluorophenylalanine; fpa, D-2-fluorophenylalanine; f₂pa,D-3,4-difluorophenylalanine; Nal, L-2-naphthylalanine; Nle, norleucine;Phg, L-α-phenylglycine; Sar, sarcosine; TNFα, tumor necrosisfactor-alpha; TNFR, TNFα receptor.

Compounds

Cyclo(FΦRRRRQ) (cFΦR₄, where Φ is L-2-naphthylalanine (SEQ ID NO.:72))was previously reported as a member of a class of cyclic CPPs (Z. Qian,et al., ACS Chem. Biol. 2013, 8, 423; Z. Qian, et al., Biochemistry2014, 53, 4034). These CPPs bind directly to the membrane phospholipids,enter cells by endocytosis, and efficiently escape from the earlyendosome into the cytosol by inducing budding of small, unstablevesicles (Id.; Z. Qian, et al., Biochemistry 2016, 55, 2601). With acytosolic delivery efficiency (defined as the ratio of cytosolic overextracellular cargo concentration) of 20%, cFΦR₄ (SEQ ID NO.:72) is anorder of magnitude more active than Tat, one of the most widely usedCPPs (Id.). Most importantly, cFΦR₄ (SEQ ID NO.:72) and other cyclicCPPs are capable of efficiently delivering a variety of cargo moleculesincluding small molecules, peptides, and proteins into the cytosol ofmammalian cells. For example, short peptidyl cargos were directlyincorporated into the cFΦR₄ (SEQ ID NO.:72) ring (endocyclic delivery)and the resulting cyclic peptides were cell-permeable (Id.; P.Upadhyaya, et al., Angew. Chem., Int. Ed. 2015, 54, 7602; Angew. Chem.2015, 127, 7712). cFΦR₄ (SEQ ID NO.:72) was also fused with 5.7 milliondifferent cyclic peptides to generate a library of cell-permeablebicyclic peptides (bicyclic delivery) (T. B. Trinh, P et al., ACS Comb.Sci. 2016, 18, 75). However, many peptide ligands must be in theirextended conformations to be biologically active and are not compatiblewith the above cyclization approaches. To this end, a reversiblecyclization strategy for intracellular delivery of linear peptidylligands was developed by fusing them with FΦR₄ (SEQ ID NO.:72) andcyclizing the fusion peptides through a disulfide bond (Z. Qian, et al.,Angew. Chem. Int. Ed. 2015, 54, 5874; Angew. Chem. 2015, 127, 5972).Unfortunately, the previous approach is limited to relatively shortpeptides, as cyclization of longer peptides results in large rings,whose conformational flexibility limits the gains in metabolic stabilityand cell-permeability (Z. Qian, et al., ACS Chem. Biol. 2013, 8, 423; Z.Qian, et al., Biochemistry 2014, 53, 4034). Cyclization via an internalthiol-containing moieties (e.g., the AA^(S) groups disclosed herein)results in smaller rings and better cellular uptake, but leaves aportion of the peptidyl cargo in the linear form, which remainssusceptible to proteolytic degradation. To overcome this limitation,disclosed herein is a reversible bicyclization strategy, which allowsthe entire CPP-cargo fusion to be converted into a bicyclic structure bythe formation of a pair of disulfide bonds (FIG. 2 ). When outside thecell, the peptide exists as a highly constrained bicycle, whichpossesses enhanced cell permeability and proteolytic stability. Uponentering the cytosol, the disulfide bonds are reduced by theintracellular glutathione (GSH) to produce the linear, biologicallyactive peptide. The bicyclic system permits the formation of a small CPPring for optimal cellular uptake and a separate cargo ring toaccommodate peptides of different lengths.

In various embodiments, the reversible bicyclic peptides describedherein comprise a first cyclic peptide sequence and a second cyclicpeptide sequence. In some embodiments, the first cycilic peptidesequence comprises a cell-penetrating sequence (X_(m)). In otherembodiments, the second cyclic peptide sequence comprises a cargopeptide sequence (X_(n)).

In some embodiments, X_(m) and X_(n) are fused. In some embodiments, thefusion occurs between the C-terminus of X_(m) and the N-terminus ofX_(n). In other embodiments, the fusion occurs between the N-terminusX_(m) and the C-terminus of X_(n). In further embodiments, an amino acidor a linking moeity may be used to fuse X_(m) and X_(n). In still otherembodiments, the amino acid or a linking moeity used to fuse X_(m) andX_(n) forms at least one intramolecular disulfide bond, thereby formingthe bicyclic peptide sequence. In further embodiments, the amino acidwhich fuses X_(m) and X_(n) is represented by AA^(S). In otherembodiments, the linking moiety may be represented by “L-J” in theformulae provided herein.

In some embodiments, X_(m) may be conjugated, directed or indirectly, toX_(n). For example, in some embodiments, X_(m) may comprise at least twoAA^(S) moeities, and X_(n) may comprise at least two AA^(S) moeities,and the X_(m) may be directly conjugated to X_(n) via two disulfidebonds formed between opposing AA^(S) residues on X_(m) and X_(n),respectively. In other embodiments, the linking moeity is covalentlybound a side chain of an amino acid in X_(m), and X_(n) comprises atleast two AA^(S) moeities, each of which form disulfide bonds with alinking moeity. Amino acids having side chains which are suitable forconjugating the linking moeity include asparagine, glutamine, aspartate,glutamate, and lysine. Further, amino acids may be appropriatelymodified for conjugation with the linking moeity.

As discussed above, to some embodiments, the bicyclic peptides describedherein comprise a linking moeity. In some embodiments, X_(m) is cyclizedthrough a linking moeity. In other embodiments, X_(n) is cyclizedthrough a linking moeity. In still other embodiments, each of X_(m) andX_(n) are independently cyclized through the linking moeity. In certainembodiments, only X_(n) is cyclized through a linking moeity, X_(m) is acyclic peptide sequence, and the linking moeity conjugates X_(n) andX_(m), thereby forming the bicyclic peptides disclosed herein.

In certain embodiments, the precursor to the linking moeity comprises atleast two thiol groups which form at least two intramolecular disulfidebonds, thereby forming the bicyclic peptides disclosed herein. In theseembodiments, the disulfide bonds can be reduced by intracellular GSH toform a linear peptide sequence comprising X_(m) and X_(n), to release alinear sequence comprising X_(m) (in which case X_(n) remains cyclic),or a linear sequence comprising X_(n) (in which case X_(m) remainscyclic). In certain embodiments after entry into the cytosol of the cellthe disulfide bonds are reduced by intracellular GSH to thereby releasea linear sequence comprising X_(n). In other embodiments, intracellularGSH reduces the two disulfide bonds to thereby release a linear sequencecomprising X_(m) and X_(n). In still other embodiments, a precursor tothe linking moiety comprises three thiol groups, which form threeintramolecular disulfide bonds in the bicyclic peptides disclosedherein. In still other embodiments, a precursor to the linking moietycomprises four thiol groups, which form four intramolecular disulfidebonds.

Accordingly, disclosed herein, in various embodiments, are bicyclicpeptides according to Formulae 1-12.

Number Formula 1

2

3

4

5

6

7

8

9

10

11

12

wherein:

-   -   X_(m) and X_(n) independently comprise a sequence of 1-50 (e.g.,        4-10) natural or non-natural amino acids, wherein X_(m)        corresponds to a cell-penetrating peptide (CPP) sequence as        defined herein and X_(n) corresponds to a cargo peptide sequence        as defined herein;    -   AA^(S) at each occurrence is independently a moiety which forms        a disulfide bond with J;    -   J is absent, or an alkyl, N-alkyl, alkenyl, alkynyl,        carbocyclyl, or heterocyclyl, each of which are independently        substituted with at least two substituents which independently        form a disulfide bond with AA^(S) at each occurrence;    -   SS at each instance represents a disulfide bond; and    -   L is absent or a moiety which links AA^(S) to an amino acid in        X_(m), X_(n), or a combination thereof.

As shown above in Formulae 1-12, X_(n) can be located on the N-terminusor C-terminus of X_(m). In some embodiments, L is present and can belinked to the N-terminus or C-terminus of X_(m), X_(n), or AA^(S). Inother embodiments, L is present and can be located between and linked toeach of X_(m) and X_(n), X_(m) and AA^(S), and/or X_(n) and AA^(S). Insome embodiments, one or more AA^(S) may be located in the X_(n). Insuch embodiments, the AA^(S) may be a component of the wild type peptidesequence (i.e., X_(n)) or AA^(S) may be introduced into the peptidesequence (X_(n)). In some embodiments, the cargo peptide sequence(X_(n)) has two AA^(S) (e.g., Formula 11), which allows for the cargopeptide sequence to be cleaved from the CPP sequence after the compoundenters the cytosol.

As used herein, “L” refers to a connection between J and X_(m), X_(n),or combinations thereof. For example, in embodiments, L may comprise amoiety which is formed between a functional group on a precursor of Jand amine group on an amino acid (e.g., on X_(m) or X_(n)). The aminegroup may be the N-terminus or it may be an amine group on a side chainof an amino acid, e.g., on X_(m). In such embodiments, the precursor ofJ can include a carboxylic acid moiety or derivative thereof (e.g., ahaloketone), which thereby forms an amide bond with the N-terminus onthe amino acid or cargo. In other embodiments, L may comprise a moietywhich is formed between a functional group on a precursor of J and acarboxylic acid group on an amino acid (e.g., on X_(m) or X_(n)). Thecarboxylic group may be the C-terminus or it may be an carboxylic acidgroup on a side chain of an amino acid, e.g., on X_(m). In suchembodiments, the precursor of J includes an amine, which thereby formsan amide bond with the C-terminus of the amino acid or the cargo.Non-limiting examples of L include at least one amino acid, alkyl,alkenyl, alkynyl, carbonyl, amide, imine, enamine, alkene, alkyne,disulfide, thioketone, sulfonylketone, carbamoyl, carbonyloxy,disulfide, thioether, and triazole. In embodiments, L is is absent, anamino acid,

As defined above, in various embodiments, J may be an alkyl, N-alkyl,alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which areindependently substituted with at least two substituents whichindependently form a disulfide bond with AA^(S) at each occurrence. Insome embodiments, J comprises at least two substituents whichindependently form a disulfide bond with AA^(S) at each occurrence. Inother embodiments, J comprises at least three substituents whichindependently form a disulfide bond with AA^(S) at each occurrence. Instill other embodiments, J comprises at least four substituents whichindependently form a disulfide bond with AA^(S) at each occurrence.

In some embodiments, L is absent, and J comprises three substituentswhich independently form a disulfide bond with AA^(S) at eachoccurrence. Examples of such a bicyclic peptide are provided in Formulae8 and 9.

In other embodiments, L is absent, and J comprises at four substituentswhich independently form a disulfide bond with AA^(S) at eachoccurrence. An example of such a bicyclic peptide is provided in Formula10.

In some embodiments, J is absent, and L links X_(m) and AA^(S).

In some embodiments, each of J and L are present. In some suchembodiments, J and L can be located on the N-terminus of X_(n) or X_(m)and can represented by at least one of the following (prior to formingdisulfide bonds):

wherein R represents the side of an amino acid which can be part of thepeptide sequence comprising X_(m) or X_(n) which can be part of L.

In other such embodiments, J and L can be located between X_(n) andX_(m), between X_(m) and X_(n), or at the C-terminus of either of X_(n)and X_(m), and can be represented by one of the following (prior toforming disulfide bonds):

To form the bicyclic peptides of the present disclosure, the hydrogon onthe thiol group of the above structures are independently replaced by abond to a sulfor group.

In some embodiments, each AA^(S) is independently an amino acid, oranalog or derivative thereof, which is capable of forming a disulfidebond (e.g., an amino acid which has a thiol group prior to forming adisulfide bond). Non-limiting examples of such amino acids, or analogsor derivatives thereof, include:

wherein the C-terminus of AA^(S) forms an amide bond or R¹, wherein R¹is OH, OR², NHR²;and wherein R² is a alkyl, aryl, heteroaryl, amino acid residue, peptidesequence of 2 to 20 amino acid residues, detectable moiety, or solidsupport.

In some embodiments, a compound of Formula 9 has the following formula:

FIG. 8 shows an embodiment of a tris-disulfide containing bicyclicpeptide according to Formula 9-A.

In some embodiments, a compound of Formula 11 has the following formula:

In some embodiments, a compound of Formula 12 has the following formula:

The Appendix attached herewith includes further illustrative formulae ofthe bicyclic peptides of the present disclosure.

In a particular aspect, disclosed herein are bicyclic peptides ofFormula I.

wherein R¹ is OH, OR², NHR², wherein R² is a C₁₋₂₀ alkyl, C₆₋₁₀ aryl orheteroaryl, amino acid residue, peptide sequence of 2 to 20 amino acidresidues, detectable moiety, or solid support; and wherein each d isindependently 1 or 2. The two peptide sequences X_(m) and X_(n) arecoupled to a central 3,5-(bismercaptomethyl)benzoyl moiety, forming abicyclic structure with a cell penetrating peptide loop of sequenceX_(m) and a cargo loop of sequence X_(n) (see FIG. 2 ). As discussedabove, X_(m) and X_(n) are used to represent peptide sequences in thebicyclic peptides described herein, and the N-terminus and C-terminus ofthe X_(m) and X_(n) may be included in various formulae provided herein(e.g., in Formula I above) to illustrate the connectivity of X_(m) andX_(n) in the bicyclic peptide. It is to be understood that when theterminal residues of X_(m) and X_(n) (e.g., —NH—, or —NH—C(O)—) areprovided in a formula, such residues do not represent additional atomsthat are required by the bicyclic peptide, but rather these residues arecomponents of the amino acids contained in X_(m) and/or X_(n). Whencompounds of Formula I are in their uncyclized form, e.g., prior toforming the disulfide bridges or after entering the cell and being actedupon by GSH, they can be represented as Formula I-A.

In an additional aspect, X_(m) and X_(n) can be coupled to the central3,5-(bismercaptomethyl)benzoyl moiety in the opposite manner than thatshown in Formula I. These compounds are also disclosed herein and arerepresented by Formula II.

Also disclosed herein are peptide sequences of Formula III.BMB-(AA^(n))_(u)   IIIwherein:

u is an integer of from 4 to 20;

each AA^(n) is, independently, a natural or non-natural amino acidresidue, with at least two AA^(n) residues independently selected fromthe group consisting of cysteine, homocysteine, an amino acid analoghaving a thiol group; and

BMB is a 3,5-bis(mercaptomethyl)benzoic acid residue.

X_(m) and X_(n)

X_(m) and X_(n) can independently comprise any suitable number of aminoacids which can be cyclized. In some embodiments, X_(m) and X_(n) canindependently comprise a sequence of from 1-50 amino acid residues(e.g., 1-20 amino acids, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 18, 19, and 20, including all ranges therebetween).In some embodiments, the combined number of amino acids in X_(m) andX_(n) is at least 8 residues. In some examples, X_(m) or X_(n) canindependently comprise 4 or more amino acid (e.g., 5 or more, 6 or more,7 or more, 8 or more, or 9 or more). In some examples, X_(m) or X_(n)can independently comprise 20 or less amino acids (e.g., 19 or less, 18or less, 17 or less, 16 or less, or 15 or less). In particularembodiments, X_(m) or X_(n) can independently comprise from 5 to 10amino acids. Each amino acid can be a natural or non-natural amino acid,or an analog or derivative thereof. Thus, the term amino acid, when usedherein, is inclusive of natural and non-natural amino acids, and analogsand derivatives thereof. The term “non-natural amino acid” refers to anorganic compound that is a congener of a natural amino acid in that ithas a structure similar to a natural amino acid so that it mimics thestructure and reactivity of a natural amino acid. The non-natural aminoacid can be a modified amino acid, and/or amino acid analog, that is notone of the 20 common naturally occurring amino acids or the rare naturalamino acids selenocysteine or pyrrolysine. Examples of suitable aminoacids include, but are not limited to, alanine, allosoleucine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, napthylalanine,phenylalanine, proline, pyroglutamic acid, serine, threonine,tryptophan, tyrosine, valine, a derivative, or combinations thereof.These are listed in the Table 1 along with their abbreviations usedherein.

TABLE 1 Amino Acid Abbreviations Abbreviations* Abbreviations* AminoAcid L-amino acid D-amino acid Alanine Ala (A) ala (a) AllosoleucineAIle aile Arginine Arg (R) arg (r) Asparagine Asn (N) asn (n) asparticacid Asp (D) asp (d) Cysteine Cys (C) cys (c) Cyclohexylalanine Cha cha2,3-diaminopropionic acid Dap dap 4-fluorophenylalanine Fpa (Σ) pfaglutamic acid Glu (E) glu (e) glutamine Gln (Q) gln (q) glycine Gly (G)gly (g) histidine His (H) his (h) Homoproline (aka pipecolic acid) Pip(Θ) pip (θ) isoleucine Ile (I) ile (i) leucine Leu (L) leu (l) lysineLys (K) lys (k) methionine Met (M) met (m) napthylalanine Nal (Φ) nal(Φ) norleucine Nle (Ω) nle phenylalanine Phe (F) phe (F) phenylglycinePhg (Ψ) phg 4- F₂Pmp (Λ) f₂pmp (phosphonodifluoromethyl) phenylalanineproline Pro (P) pro (p) sarcosine Sar (Ξ ) sar selenocysteine Sec (U)sec (u) serine Ser (S) ser (s) threonine Thr (T) thr (y) tyrosine Tyr(Y) tyr (y) tryptophan Trp (W) trp (w) Valine Val (V) val (v)2,3-diaminopropionic acid Dap dap *single letter abbreviations: whenshown in capital letters herein it indicates the L-amino acid form, whenshown in lower case herein it indicates the D-amino acid form

As discussed above, non-natural amino acids and D-amino acids can beused herein. The disclosed methods and compositions are particularlywell suited for incorporating non-natural and D-amino acids. The aminoacids can be coupled by a peptide bond. Each amino acids can be coupledto an adjacent amino acid at the amino group, the carboxylate group, orthe side chain.

X_(m)

The amino acid sequence X_(m) can be a cell penetrating peptidesequence. In some embodiments, X_(m) is from 4 to 20 (e.g., 5 to 10)amino acid residues in length. In some embodiments, at least one, atleast two, or at least three amino acids in X_(m) are arginine. In someexamples, at least one, at least two, or at least three amino acids inX_(m) have a hydrophobic side chain. Non-limiting examples of aminoacids having a hydrophopbic side chain include glycine, alanine, valine,leucine, isoleucine, methionine, phenylalanine, tryptophan, proline,naphthylalanine, phenylglycine, homophenylalanine, tyrosine,cyclohexylalanine, or norleucine. In particular embodiments, thehydrophobic side chain is a hydrophobic aromatic aide chain. In someembodiments, amino acids having an aromatic hydrophobic side chaininclude naphthylalanine, phenylglycine, homophenylalanine,phenylalanine, tryptophan, and tyrosine. In particular embodiments, theamino acid having a hydrophobic is phenylalanine, naphthylalanine,tryptophan, or an analog or derivative thereof. In some examples, atleast one amino acid in X_(m) comprises phenylalanine, phenylglycine, orhistidine, or analogs or derivatives thereof. In some examples, X_(m)comprises at least one arginine or an analog or derivative thereof.

In some specific examples of Formula I or II, X_(m) can independentlyselected from any of the sequences listed in Table 2. In some examples,the cell penetrating peptide can be the reverse of any of the sequenceslisted in Table 2.

TABLE 2 Example sequences for X_(m). SEQ ID NO CPP sequence 64 FΦRRR 65FΦRRRC 66 FΦRRRU 67 RRRΦF 68 RRRRΦF 69 FΦRRRR 70 FϕRrR 71 FϕRrR 72FΦRRRR 73 fORrRr 74 RRFRΦR 75 FRRRRΦ 76 rRFRΦR 77 RRΦFRR 78 CRRRRFW 79FfΦRrRr 80 FFΦRRRR 81 RFRFRΦR 82 URRRRFW 83 CRRRRFW 84 FΦRRRRQK 85FΦRRRRQC 86 fΦRrRrR 87 FΦRRRRR 88 RRRRΦFDΩC 89 FΦRRR 90 FWRRR 91 RRRΦF92 RRRWF 93 FΦRRRR 94 FFRRR 95 FFrRr 96 FFRrR 97 FRFRR 98 FRRFR 99 FRRRF100 GΦRRR 101 FFFRA 102 FFFRR 103 FFRRRR 104 FRRFRR 105 FRRRFR 106RFFRRR 107 RFRRFR 108 FRFRRR 109 FFFRRR 110 FFRRRF 111 FRFFRR 112 RRFFFR113 FFRFRR 114 FFRRFR 115 FRRFFR 116 FRRFRF 117 FRFRFR 118 RFFRFR 119GΦRRRR 120 FFFRRRR 121 RFFRRRR 122 RRFFRRR 123 RFFFRRR 124 RRFFFRR 125FFRRFRR 126 FFRRRRF 127 FRRFFRR 128 FFFRRRRR 129 FFFRRRRRR 130 FΦRrRr131 XXRRRR 132 FfFRrR 133 fFfrRr 134 fFfRrR 135 FfFrRr 136 fFφrRr 137fΦfrRr 138 φFfrRr 139 FΦrRr 140 fΦrRr 141 Ac-Lys-fFRrRrD 142Ac-Dap-fFRrRrD 143 WWWRRRR 144 WWWRRRRR 145 FWRRRR 146 WWWRRRΦ = L-naphthylalanine; ϕ = D-naphthylalanine; Ω = L-norleucine; r= D-arginine; F = L-phenylalanine; f = D-phenylalanine; q = D-glutamine;X = L-4-fluorophenylalanine; Dap = L-2,3-diaminopropionic acid.

In some examples, X_(m) can by any of SEQ ID NO:64 to SEQ ID NO:146. Insome examples, X_(m) can be a variant of any of SEQ ID NO:64 to SEQ IDNO:146. Also disclosed herein are cyclic sequences of the peptides inTable 2. Sequences 64-146 can also be modified by having one or morecysteine residues (or other amino acid having a thiol group) internallyor at one or both ends (i.e., at the C- and/or N-terminus), which allowsfor cyclization of the peptide by forming a disulfide bond with thecysteine (or other amino acid having a thiol group).

Peptide variants are well understood to those of skill in the art andcan involve amino acid sequence modifications. For example, amino acidsequence modifications typically fall into one or more of three classes:substitutional, insertional, or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of 1 to 3 residues.Deletions are characterized by the removal of one or more amino acidresidues from the peptide sequence. Typically, no more than from 1 to 3residues are deleted at any one site within the peptide. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 3 amino acid residues; and deletions will rangeabout from 1 to 3 residues. Deletions or insertions preferably are madein adjacent pairs, i.e. a deletion of 2 residues or insertion of 2residues. Substitutions, deletions, insertions or any combinationthereof can be combined to arrive at a final construct. Substitutionalvariants are those in which at least one residue has been removed and adifferent residue inserted in its place. Such substitutions generallyare made in accordance with the following Table 3 and are referred to asconservative substitutions.

TABLE 3 Amino Acid Substitutions Exemplary Conservative SubstitutionsAla replaced by Ser Arg replaced by Lys or Gln Asn replaced by Gln orHis Asp replaced by Glu Cys replaced by Ser Gln replaced by Asn or LysGlu replaced by Asp Gly replaced by Pro His replaced by Asn or Gln Ilereplaced by Leu or Val Leu replaced by Ile or Val Lys replaced by Arg orGln Met replaced by Leu or Ile Phe replaced by Met, Leu, Nal, Phg, orTyr Ser replaced by Thr Thr replaced by Ser Trp replaced by Tyr Tyrreplaced by Trp or Phe Val replaced by Ile or Leu

Substantial changes in function are made by selecting substitutions thatare less conservative than those in Table 3, i.e., selecting residuesthat differ more significantly in their effect on maintaining (a) thestructure of the peptide backbone in the area of the substitution, forexample as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site or (c) the bulk of theside chain. The substitutions which in general are expected to producethe greatest changes in the protein properties will be those in which(a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for(or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl,valyl or alanyl; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g., lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine, in this case, (e) byincreasing the number of sites for sulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the peptidesprovided herein.

It is understood that one way to define the variants of X_(m) is throughdefining the variants in terms of homology/identity to specific knownsequences. For example, SEQ ID NO:64 to SEQ ID NO:146 each sets forth aparticular sequence. Specifically disclosed are variants of thesepeptide that have at least, 85%, 90%, 95%, 97%, or 99% homology to SEQID NO:64 to SEQ ID NO:146. Those of skill in the art readily understandhow to determine the homology of two proteins. For example, the homologycan be calculated after aligning the two sequences so that the homologyis at its highest level.

In addition to variants of SEQ ID NO:64 to SEQ ID NO:146 are derivativesof these peptides which also function in the disclosed methods andcompositions. Derivatives are formed by replacing one or more residueswith a modified residue, where the side chain of the residue has beenmodified.

In particular examples, X_(m) comprises at least one, at least two, ormore specifically, at least three adjacent arginine (R or r) residues.Further, in these structures there are at least one, at least two, or atleast three hydrophobic residues, for example, phenylalanine,naphthylalanine, tryptophan, or an analog or derivative thereof. Forexample, there can be 1 arginine and 5 hydrophobic residues likephenylalanine, naphthylalanine, tryptophan, or an analog or derivativethereof, 2 arginine and 4 hydrophobic residues like phenylalanine,naphthylalanine, tryptophan, or an analog or derivative thereof, 3arginine and 3 hydrophobic residues like phenylalanine, naphthylalanine,tryptophan, or an analog or derivative thereof, 4 arginine and 2hydrophobic residues like phenylalanine, naphthylalanine, tryptophan, oran analog or derivative thereof, or 4 arginine and 1 hydrophobic residuelike phenylalanine, naphthylalanine, tryptophan, or an analog orderivative thereof. In a specific example, the cyclic compoundsdisclosed herein have 3 arginies and 3 hydrophobic residues likephenylalanine, naphthylalanine, tryptophan, or an analog or derivativethereof. Further the arginine residues can be clustered, e.g., anarginine is within 2 amino acids of another arginine residue. Likewise,the hydrophobic residues can be clustered, e.g., one hydrophobic residueis with 2 amino acids of another hydrophobic residue.

In a preferred example, X_(m) is or comprises RRRRΦF, FΦRRRR, FfΦRrRr,fΦRrRr, fΦRrRr, RφrRrR, or RφrRrR.

In some embodiments, the amino acid sequence X_(m) can be represented asor can compriseAA¹-AA²-AA³-AA⁴-AA⁵-(AA⁶)_(m)-(AA⁷)_(n)-(AA⁸)_(p)-(AA⁹)_(q)wherein:

-   -   AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹ are each        independently an amino acid;    -   at least three amino acids are arginine;    -   at least two amino acids comprise a hydrophobic side chain;    -   m, n, p, or q are independently selected from 0 and 1.

In some embodiments, AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹corresponds to at least one amino acid sequence of SEQ ID NO:64 to SEQID NO:146.

Certain embodiments of the invention include amino acid sequenceswherein at least four consecutive amino acids have alternatingchirality. As used herein, chirality refers to the “D” and “L” isomersof amino acids. In particular embodiments of the invention, at leastfour consecutive amino acids have alternating chirality and theremaining amino acids are L-amino acids. In other embodiments, thepeptides of the invention comprise a four amino acid sequence havingD-L-D-L chirality. In still other embodiments, the peptides of theinvention comprise a four amino acid sequence having L-D-L-D chirality.

In embodiments, peptides of the invention comprise two consecutiveL-amino acids. In further embodiments, peptides of the inventioncomprise two consecutive L-amino acids separating two D-amino acids. Inyet further embodiments, peptides of the invention comprise twoconsecutive L-amino acids separating two D-amino acids and at least fourconsecutive amino acids having alternating chirality, such as, but notlimited to peptide sequences with D-L-L-D-L-D or L-D-L-L-D-L-Dchirality. In even further embodiments, peptides of the inventioncomprise two consecutive L-amino acids separating two D-amino acids andat least five consecutive amino acid having alternating chirality, suchas, but not limited to peptide sequences with D-L-L-D-L-D-L orL-D-L-L-D-L-D-L chirality.

In embodiments, peptides of the invention comprise two consecutiveD-amino acids. In further embodiments, peptides of the inventioncomprise two consecutive D-amino acids separating two L-amino acids. Instill further embodiments of the invention, peptides of the inventioncomprise two consecutive D-amino acids separating two L-amino acids andat least four consecutive amino acids having alternating chirality, suchas, but not limited to peptide sequences with L-D-D-L-D-L. In evenfurther embodiments of the invention, peptides of the invention comprisetwo consecutive D-amino acids separating two L-amino acids and at leastfive consecutive amino acids having alternating chirality, such as, butnot limited to peptide sequences with L-D-D-L-D-L-D.

In some embodiments, the amino acid sequence with alternating chiralitycomprises about at least about 4 amino acids, at least about 5 aminoacids, at least about 6 amino acids, at least about 7 amino acids, atleast about 8 amino acids or at least about 9 amino acids. Inembodiments, the amino acid sequence with alternating chiralitycomprises of from about 4 amino acids to about 9 amino acids, or about 5amino acids to about 6 amino acids, or about 7 amino acids to about 9amino acids, or about 8 amino acids to about 9 amino acids, or about 4amino acids to about 8 amino acids, or about 4 amino acids to about 7amino acids, or about 4 amino acids to about 6 amino acids, or about 4amino acids to about 5 amino acids.

In certain embodiments, the peptides of the invention comprise at leastone hydrophobic residue. In further embodiments, the peptides of theinvention comprise two hydrophobic residues. In still furtherembodiments, the peptides of the invention comprise at least twohydrophobic residues. In certain embodiments, at least one hydrophobicresidue is an aromatic hydrophobic residue. In particular embodiments,at least one hydrophobic residue is selected from the group consistingof naphthylalanine, phenylalanine, tryptophan, and tyrosine. In furtherembodiments, at least one hydrophobic residue is selected from the groupconsisting of naphthylalanine and phenylalanine. In certain embodiments,peptides of the invention comprise at least one naphthylalanine. In yetother embodiments, peptides of the invention comprise at least onephenylalanine. In still other embodiments, peptides of the inventioncomprise at least one phenylalanine and at least one naphthylalanine. Incertain embodiments of the invention, the peptide comprises at least onehydrophobic residue in the AA¹, AA², or AA³ position. In certainembodiments, the peptide comprises at least one aromatic hydrophobicresidue in the AA¹, AA², or AA³ position. In further embodiments of theinvention, the peptide comprises at least one hydrophobic residueselected from the group consisting of naphthylalanine and phenylalaninein the AA¹, AA², or AA³ position.

In certain aspects, disclosed herein are bicyclic peptides of Formula V,VI, VII, VIII, IX, X, and XII:

or a pharmaceutically acceptable salt thereof, wherein AA¹, AA², AA³,AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, AA⁹, AA¹⁰, and AA¹¹ are each independently anamino acid.

In some embodiments, at least three amino acids are arginine. In furtherembodiments, at least two amino acids comprise a hydrophobic residue. Insome embodiments, AA^(S) at each occurrence is independently a moietywhich forms a disulfide bond with J.

In some embodiments, J is N-alkyl, aryl, or heteroaryl, each of whichare independently substituted with at least two substituents whichindependently form a disulfide bond with AA^(S) at each occurrence. Insome embodiments J is selected from the group consisting of

In some embodiments, L is selected from the group consisting of

In some embodiments, the compound is selected from the group consistingof:

or pharmaceutically acceptable salt thereof, wherein each d isindependently 1 or 2.

When compounds of Formula V, VI, VII, VIII, IX, or X are in theiruncyclized form, e.g., prior to forming the disulfide bridges or afterentering the cell and being acted upon by GSH, they can be representedas Formula I-A. Also disclosed are compounds according to Formula V-A,VI-A, VII-A, VIII-A, IX-A, and X-A:

or a pharmaceutically acceptable salt thereof,wherein:

-   -   AA^(S)′ at each occurrence is independently a moiety which        comprises a thiol;    -   J′ is an alkyl, N-alkyl, alkenyl, alkynyl, carbocyclyl, or        heterocyclyl, each of which are independently substituted with        at least two thiol substituents; and    -   X_(n) and L are defined herein.

In some embodiments, J is N-alkyl or aryl. In some embodiments, J′ is

In some embodiments, each AA^(S) is independently:

wherein the C-terminus of AA^(S)′ forms an amide bond or is R¹, whereinR¹ is OH, OR², NHR²;and wherein R² is a alkyl, aryl, heteroaryl, amino acid, peptidesequence of 2 to 20 amino acid, detectable moiety, or solid support.

In some embodiments, the compound has a structure selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.

X_(n)

The amino acid sequence X_(n) is a cargo peptide sequence. As discussedabove, there is no limitation on the number of amino acids X_(n). Insome embodiments, X_(n) may have from 1 to 50 amino acids, e.g., from 1to 20, from 1 to 10, from 4 to 20, from 4 to 10, and all values andsubrages therein. Examples of sequences for X_(n) are those outlinedabove in Table 2 including variants or derivatives thereof. Additionalexamples of cargo peptide sequences can comprise any of those listed inTable 4 or Table 5, or derivatives or combinations thereof.

TABLE 4 Example Cargo Sequences SEQ ID NO Sequence 1 HKGFY 2 AFWTG 3HALSΩ 4 ΨYAKYFGKH-Dap 5 AFWTEKΩLAH-Dap 6 F-Dap-SVPYH-Dap 7 WFDKFNH-Dap 8dΦ-SQ-dΦ-KFRVR-Dap 9 RRdΦ-R-fF-KFQG-Dap 10 OR-dΦ-R-fF-KFQG-Dap 11 RFZZFK12 RDΨZNK 13 ZZPGAK 14 ZZASAK 15 ZZLPTK 16 ΨRNZIK 17 ZTEANK 18 Z-dΦ-VGQK19 ZΨSZZK 20 ZΨMSZK 21 ZSMZGK 22 ZSΨZZK 23 ZRVDAK 24 RDΨPra-N 25ΦRRRR-Dap 26 ΨRN-Pra-I 27 Pra-SΨKK 28 Pra-RVDA 29 AΨRN-Pra-I 30ΨRN-Pra-IA 31 AΨRN-Pra-IA 32 AAΨRN-Pra-IA 33 AFΨRN-Pra-I-A 34A-AbuΨRN-Pra-I-Abu 35 ΨIΨRN-Pra-I-Abu-K 36 ΨΨRN-Pra-I-Abu 37ALΨRN-Pra-ID 38 AQΨRN-Pra-ID 39 IEΨRN-Pra-ID 40 ASΨRN-Pra-IE 41LΨPRN-Pra-IE 42 AΨΨRN-Pra-IF 43 A-OmΨRN-Pra-IF 44 A-AbuΨRN-Pra-IN 45dA-AΨRN-Pra-IN 46 ΨNΨRN-Pra-II 47 A-AbuΨRN-Pra-I-Nle 48 WΨRN-Pra-IΨ 49ANΨRN-Pra-IR 50 R-ΩΨRN-Pra-IS 51 HΨRN-Pra-IYK-Φ 52 A-AbuΨRN-Pra-I-Abu 53ΨIΨRN-Pra-I-Abu 54 ALΨRN-Pra-ID 55 AQΨRN-Pra-ID 56 A-OrnΨRN-Pra-IF 57AΨΨRN-Pra-IF 58 A-AbuΨRN-Pra-I-Abu 59 AAΨRN-Pra-IA 60 AAFRN-Pra-IA 61ALFRN-Pra-ID 62 ΨYAKYFGKH 63 AFWTEKΩLAH

TABLE 5 Example cargo moieties SEQ ID NO Abbreviation Sequence* 147 R₅RRRRR 148 A₅ AAAAA 149 F₄ FFFF 150 PCP DE(pCAP)LI 151 A₇ AAAAAAA 152RARAR 153 DADAD 154 DΩUD 155 UTRV 156 SASAS 157 ALDWSWLQ 158 ALDASALQ159 SFAEYWALLS *pCAP, phosphocoumaryl amino propionic acid; Ω,norleucine; U, 2-aminobutyric acid.

It should be understood that when referring to Formula I, the sequenceX_(n) is coupled to a cysteine residue (C) at each end, and the amidebonds formed by said coupling are included in Formula I. Likewise, whenreferring to Formula II, X_(m) is coupled to a cystein residue at eachend, and the amide bonds formed by said coupling are included in FormulaII. For example, these sections can be represented as —C—X_(n)—C— or—C—X_(m)—C—, or sometimes as —C-(AA)_(n)C—, where AA= an amino acidresidue as defined herein and n is an integer of from 2 to 8, e.g.,—C-AA¹-AA²-C—, —C-AA¹-AA²-AA³-C—, —C-AA¹-AA²-AA³-AA⁴-C—,—C-AA′-AA²-AA³-AA⁴-AA⁵-C—, —C-AA¹-AA²-AA³-AA⁵-AA⁶-C—,—C-AA¹-AA²-AA³-AA⁵-AA⁶-AA⁷-C—, and —C-AA¹-AA²-AA³-AA⁵-AA⁶-AA⁷-AA⁸-C—.When referring to Formula I, the two terminal cystein residues attachedto sequence X_(n) are coupled to the 3,5-(bismercaptomethylbenzoyl)moeity of Formula I and one cycteine residue is also coupled to sequenceX_(m) (which is also coupled to the 3,5-(bismercaptomethylbenzoyl)moiety of Formula I). When referring to Formula II, the two terminalcystein residues attached to sequence X_(m) are coupled to the3,5-(bismercaptomethylbenzoyl) moiety of Formula II and one cycteinresidue is also coupled to sequence X_(n) (which is also coupled to the3,5-(bismercaptomethylbenzoyl) moiety of Formula II).

SPECIFIC EXAMPLES

Specific examples of bicyclic peptides disclosed herein are shown inTable 6.

TABLE 6 Sequences of peptides in this work^(a) Peptide SEQ ID NO^(b)Sequence 1 160 RQIKIWFQNRRMKWKKGG-TALDWSWLQTE 2 161

3 162

4 163

5 164

6 165

7 166

8 167

^(a)BMB, 3,5-bis(mercaptomethyl)benzoyl; Φ, L-2-naphthylalanine; MP,3-mercaptopropionyl. ^(b)underlined portion only.

Detectable Moiety

The disclosed compounds can also comprise a detectable moiety, e.g.,linked to a side chain of any amino acid in X_(m) or X_(n) or on R¹. Thedetectable moiety can comprise any detectable label. Examples ofsuitable detectable labels include, but are not limited to, a UV-Vislabel, a near-infrared label, a luminescent group, a phosphorescentgroup, a magnetic spin resonance label, a photosensitizer, aphotocleavable moiety, a chelating center, a heavy atom, a radioactiveisotope, a isotope detectable spin resonance label, a paramagneticmoiety, a chromophore, or any combination thereof. In some embodiments,the label is detectable without the addition of further reagents.

In some embodiments, the detectable moiety is a biocompatible detectablemoiety, such that the compounds can be suitable for use in a variety ofbiological applications. “Biocompatible” and “biologically compatible”,as used herein, generally refer to compounds that are, along with anymetabolites or degradation products thereof, generally non-toxic tocells and tissues, and which do not cause any significant adverseeffects to cells and tissues when cells and tissues are incubated (e.g.,cultured) in their presence.

The detectable moiety can contain a luminophore such as a fluorescentlabel or near-infrared label. Examples of suitable luminophores include,but are not limited to, metal porphyrins; benzoporphyrins;azabenzoporphyrine; napthoporphyrin; phthalocyanine; polycyclic aromatichydrocarbons such as perylene, perylene diimine, pyrenes; azo dyes;xanthene dyes; boron dipyoromethene, aza-boron dipyoromethene, cyaninedyes, metal-ligand complex such as bipyridine, bipyridyls,phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium;acridine, oxazine derivatives such as benzophenoxazine; aza-annulene,squaraine; 8-hydroxyquinoline, polymethines, luminescent producingnanoparticle, such as quantum dots, nanocrystals; carbostyril; terbiumcomplex; inorganic phosphor; ionophore such as crown ethers affiliatedor derivatized dyes; or combinations thereof. Specific examples ofsuitable luminophores include, but are not limited to, Pd (II)octaethylporphyrin; Pt (II)-octaethylporphyrin; Pd (II)tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II)meso-tetraphenylporphyrin tetrabenzoporphine; Pt (II) meso-tetraphenymetrylbenzoporphyrin; Pd (II) octaethylporphyrin ketone; Pt (II)octaethylporphyrin ketone; Pd (II)meso-tetra(pentafluorophenyl)porphyrin; Pt (II) meso-tetra(pentafluorophenyl) porphyrin; Ru (II)tris(4,7-diphenyl-1,10-phenanthroline) (Ru (dpp)₃); Ru (II)tris(1,10-phenanthroline) (Ru(phen)₃), tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)₃); erythrosine B; fluorescein;fluorescein isothiocyanate (FITC); eosin; iridium (III)((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)); indium (III)((benzothiazol-2-yl)-7-(diethylamino)-coumarin))-2-(acetylacetonate);Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow;Texas Red; rhodamine B; rhodamine 6G; sulfur rhodamine; m-cresol; thymolblue; xylenol blue; cresol red; chlorophenol blue; bromocresol green;bromcresol red; bromothymol blue; Cy2; a Cy3; a Cy5; a Cy5.5; Cy7;4-nitirophenol; alizarin; phenolphthalein; o-cresolphthalein;chlorophenol red; calmagite; bromo-xylenol; phenol red; neutral red;nitrazine; 3,4,5,6-tetrabromphenolphtalein; congo red; fluorescein;eosin; 2′,7′-dichlorofluorescein; 5(6)-carboxy-fluorecsein;carboxynaphthofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid;semi-naphthorhodafluor; semi-naphthofluorescein; tris(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride;(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) tetraphenylboron;platinum (II) octaethylporphyin; dialkylcarbocyanine;dioctadecylcycloxacarbocyanine; fluorenylmethyloxycarbonyl chloride;7-amino-4-methylcourmarin (Amc); green fluorescent protein (GFP); andderivatives or combinations thereof.

In some examples, the detectable moiety can comprise Rhodamine B (Rho),fluorescein isothiocyanate (FITC), 7-amino-4-methylcourmarin (Amc),green fluorescent protein (GFP), or derivatives or combinations thereof.

The detectible moiety can be attached to the cell penetrating peptidemoiety at the amino group, the carboxylate group, or the side chain ofany of the amino acids of the cell penetrating peptide moiety or cargomoiety (e.g., at the amino group, the carboxylate group, or the sidechain or any of X_(m) or X_(n) or R¹).

Therapeutic Moiety

The disclosed compounds can also comprise a therapeutic moiety. In someexamples, the cargo moiety comprises a therapeutic moiety. Thedetectable moiety can be linked to a therapeutic moiety or thedetectable moiety can also serve as the therapeutic moiety. Therapeuticmoiety refers to a group that when administered to a subject will reduceone or more symptoms of a disease or disorder.

The therapeutic moiety can comprise a wide variety of drugs, includingantagonists, for example enzyme inhibitors, and agonists, for example atranscription factor which results in an increase in the expression of adesirable gene product (although as will be appreciated by those in theart, antagonistic transcription factors can also be used), are allincluded. In addition, therapeutic moiety includes those agents capableof direct toxicity and/or capable of inducing toxicity towards healthyand/or unhealthy cells in the body. Also, the therapeutic moiety can becapable of inducing and/or priming the immune system against potentialpathogens.

The therapeutic moiety can, for example, comprise an anticancer agent,antiviral agent, antimicrobial agent, anti-inflammatory agent,immunosuppressive agent, anesthetics, or any combination thereof.

The therapeutic moiety can comprise an anticancer agent. Exampleanticancer agents include 13-cis-Retinoic Acid,2-Amino-6-Mercaptopurine, 2-CdA, 2-Chlorodeoxyadenosine, 5-fluorouracil,6-Thioguanine, 6-Mercaptopurine, Accutane, Actinomycin-D, Adriamycin,Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin,Alkaban-AQ, Alkeran, All-transretinoic acid, Alpha interferon,Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide,Anandron, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Arimidex,Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU,Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin,Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath,Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin,Carmustine, Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine,cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine,Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine,Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin,Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride,Daunorubicin liposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone,Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate,Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC,Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposomal, Droxia,DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar,Emcyt, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase,Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate,Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim,Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil,Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR,Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin,Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex,Mechlorethamine, -Mechlorethamine Hydrochlorine, Medralone, Medrol,Megace, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna,Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel,Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide,Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate,Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone,Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred,PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON,PEG-L-asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ,Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin,Prolifeprospan 20 with Carmustine implant, Purinethol, Raloxifene,Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alfa-2a), Rubex,Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim,Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin,Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide,Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide,Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab,Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid,Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs,Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon,Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa,Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulatingfactor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine,HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisonesodium phosphate, Hydrocortisone sodium succinate, Hydrocortonephosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin,Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate,Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEGconjugate), Interleukin 2, Interleukin-11, Intron A (interferonalfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine,Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L-PAM, L-Sarcolysin,Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX,Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan,Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. Thetherapeutic moiety can also comprise a biopharmaceutical such as, forexample, an antibody.

In some examples, the therapeutic moiety can comprise an antiviralagent, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc.

In some examples, the therapeutic moiety can comprise an antibacterialagent, such as acedapsone; acetosulfone sodium; alamecin; alexidine;amdinocillin; amdinocillin pivoxil; amicycline; amifloxacin; amifloxacinmesylate; amikacin; amikacin sulfate; aminosalicylic acid;aminosalicylate sodium; amoxicillin; amphomycin; ampicillin; ampicillinsodium; apalcillin sodium; apramycin; aspartocin; astromicin sulfate;avilamycin; avoparcin; azithromycin; azlocillin; azlocillin sodium;bacampicillin hydrochloride; bacitracin; bacitracin methylenedisalicylate; bacitracin zinc; bambermycins; benzoylpas calcium;berythromycin; betamicin sulfate; biapenem; biniramycin; biphenaminehydrochloride; bispyrithione magsulfex; butikacin; butirosin sulfate;capreomycin sulfate; carbadox; carbenicillin disodium; carbenicillinindanyl sodium; carbenicillin phenyl sodium; carbenicillin potassium;carumonam sodium; cefaclor; cefadroxil; cefamandole; cefamandole nafate;cefamandole sodium; cefaparole; cefatrizine; cefazaflur sodium;cefazolin; cefazolin sodium; cefbuperazone; cefdinir; cefepime; cefepimehydrochloride; cefetecol; cefixime; cefmenoxime hydrochloride;cefmetazole; cefmetazole sodium; cefonicid monosodium; cefonicid sodium;cefoperazone sodium; ceforanide; cefotaxime sodium; cefotetan; cefotetandisodium; cefotiam hydrochloride; cefoxitin; cefoxitin sodium;cefpimizole; cefpimizole sodium; cefpiramide; cefpiramide sodium;cefpirome sulfate; cefpodoxime proxetil; cefprozil; cefroxadine;cefsulodin sodium; ceftazidime; ceftibuten; ceftizoxime sodium;ceftriaxone sodium; cefuroxime; cefuroxime axetil; cefuroxime pivoxetil;cefuroxime sodium; cephacetrile sodium; cephalexin; cephalexinhydrochloride; cephaloglycin; cephaloridine; cephalothin sodium;cephapirin sodium; cephradine; cetocycline hydrochloride; cetophenicol;chloramphenicol; chloramphenicol palmitate; chloramphenicol pantothenatecomplex; chloramphenicol sodium succinate; chlorhexidine phosphanilate;chloroxylenol; chlortetracycline bisulfate; chlortetracyclinehydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin hydrochloride;cirolemycin; clarithromycin; clinafloxacin hydrochloride; clindamycin;clindamycin hydrochloride; clindamycin palmitate hydrochloride;clindamycin phosphate; clofazimine; cloxacillin benzathine; cloxacillinsodium; cloxyquin; colistimethate sodium; colistin sulfate; coumermycin;coumermycin sodium; cyclacillin; cycloserine; dalfopristin; dapsone;daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline;denofungin; diaveridine; dicloxacillin; dicloxacillin sodium;dihydrostreptomycin sulfate; dipyrithione; dirithromycin; doxycycline;doxycycline calcium; doxycycline fosfatex; doxycycline hyclate; droxacinsodium; enoxacin; epicillin; epitetracycline hydrochloride;erythromycin; erythromycin acistrate; erythromycin estolate;erythromycin ethylsuccinate; erythromycin gluceptate; erythromycinlactobionate; erythromycin propionate; erythromycin stearate; ethambutolhydrochloride; ethionamide; fleroxacin; floxacillin; fludalanine;flumequine; fosfomycin; fosfomycin tromethamine; fumoxicillin;furazolium chloride; furazolium tartrate; fusidate sodium; fusidic acid;gentamicin sulfate; gloximonam; gramicidin; haloprogin; hetacillin;hetacillin potassium; hexedine; ibafloxacin; imipenem; isoconazole;isepamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin;levofuraltadone; levopropylcillin potassium; lexithromycin; lincomycin;lincomycin hydrochloride; lomefloxacin; Lomefloxacin hydrochloride;lomefloxacin mesylate; loracarbef; mafenide; meclocycline; meclocyclinesulfosalicylate; megalomicin potassium phosphate; mequidox; meropenem;methacycline; methacycline hydrochloride; methenamine; methenaminehippurate; methenamine mandelate; methicillin sodium; metioprim;metronidazole hydrochloride; metronidazole phosphate; mezlocillin;mezlocillin sodium; minocycline; minocycline hydrochloride; mirincamycinhydrochloride; monensin; monensin sodiumr; nafcillin sodium; nalidixatesodium; nalidixic acid; natainycin; nebramycin; neomycin palmitate;neomycin sulfate; neomycin undecylenate; netilmicin sulfate;neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone;nifurdazil; nifurimide; nifiupirinol; nifurquinazol; nifurthiazole;nitrocycline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium;ofloxacin; onnetoprim; oxacillin; oxacillin sodium; oximonam; oximonamsodium; oxolinic acid; oxytetracycline; oxytetracycline calcium;oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin;pefloxacin; pefloxacin mesylate; penamecillin; penicillin G benzathine;penicillin G potassium; penicillin G procaine; penicillin G sodium;penicillin V; penicillin V benzathine; penicillin V hydrabamine;penicillin V potassium; pentizidone sodium; phenyl aminosalicylate;piperacillin sodium; pirbenicillin sodium; piridicillin sodium;pirlimycin hydrochloride; pivampicillin hydrochloride; pivampicillinpamoate; pivampicillin probenate; polymyxin B sulfate; porfiromycin;propikacin; pyrazinamide; pyrithione zinc; quindecamine acetate;quinupristin; racephenicol; ramoplanin; ranimycin; relomycin;repromicin; rifabutin; rifametane; rifamexil; rifamide; rifampin;rifapentine; rifaximin; rolitetracycline; rolitetracycline nitrate;rosaramicin; rosaramicin butyrate; rosaramicin propionate; rosaramicinsodium phosphate; rosaramicin stearate; rosoxacin; roxarsone;roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin;sarpicillin; scopafungin; sisomicin; sisomicin sulfate; sparfloxacin;spectinomycin hydrochloride; spiramycin; stallimycin hydrochloride;steffimycin; streptomycin sulfate; streptonicozid; sulfabenz;sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfacytine;sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfalene;sulfamerazine; sulfameter; sulfamethazine; sulfamethizole;sulfamethoxazole; sulfamonomethoxine; sulfamoxole; sulfanilate zinc;sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet;sulfisoxazole; sulfisoxazole acetyl; sulfisboxazole diolamine;sulfomyxin; sulopenem; sultamricillin; suncillin sodium; talampicillinhydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin;tetracycline; tetracycline hydrochloride; tetracycline phosphatecomplex; tetroxoprim; thiamphenicol; thiphencillin potassium;ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium;ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate;tosufloxacin; trimethoprim; trimethoprim sulfate; trisulfapyrimidines;troleandomycin; trospectomycin sulfate; tyrothricin; vancomycin;vancomycin hydrochloride; virginiamycin; or zorbamycin.

In some examples, the therapeutic moiety can comprise ananti-inflammatory agent.

In some examples, the therapeutic moiety can comprise dexamethasone(Dex).

In other examples, the therapeutic moiety comprises a therapeuticprotein. For example, some people have defects in certain enzymes (e.g.,lysosomal storage disease). It is disclosed herein to deliver suchenzymes/proteins to human cells by linking to the enzyme/protein to oneof the disclosed cell penetrating peptides. The disclosed cellpenetrating peptides have been tested with proteins (e.g., GFP, PTP1B,actin, calmodulin, troponin C) and shown to work.

Targeting Moieties

The disclosed compounds can also comprise a targeting moiety. In someexamples, the cargo moiety comprises a targeting moiety. The targetingmoiety can comprise, for example, a sequence of amino acids that cantarget one or more enzyme domains. In some examples, the targetingmoiety can comprise an inhibitor against an enzyme that can play a rolein a disease, such as cancer, cystic fibrosis, diabetes, obesity, orcombinations thereof. For example, the targeting moiety can comprise anyof the sequences listed in Table 7.

TABLE 7 Example targeting moieties SEQ ID NO Abbreviation * Sequence 168PΘGΛYR Pro-Pip-Gly-F₂Pmp-Tyr- 169 SΘIΛΛR Ser-Pip-Ile-F₂Pmp-F₂Pmp- 170IHIΛIR Ile-His-Ile-F₂Pmp-Ile- 171 AaIΛΘR Ala-(D-Ala)-Ile-F₂Pmp-Pip- 172ΣSΘΛvR Fpa-Ser-Pip-F₂Pmp-(D-Val)- 173 ΘnPΛAR Pip-(D-Asn)-Pro-F₂Pmp-Ala-174 TΨAΛGR Tyr-Phg-Ala-F₂Pmp-G1y- 175 AHIΛaR Ala-His-Ile- F₂Pmp-(D-Ala)-176 GnGΛpR Gly-(D-Asn)-Gly-F₂Pmp-(D-Pro)- 177 fQΘΛIR(D-Phe)-Gln-Pip-F₂Pmp-Ile- 178 SPGΛHR Ser-Pro-Gly-F₂Pmp-His- 179 ΘYIΛHRPip-Tyr-Ile-F₂Pmp-His- 180 SvPΛHR Ser-(D-Val)-Pro-F₂Pmp-His- 181 AIPΛnRAla-Ile-Pro-F₂Pmp-(D-Asn)- 182 ΣSIΛQF Fpa-Ser-Ile-F₂Pmp-Gln- 183 AaΨΛfRAla-(D-Ala)-Phg-F₂Pmp-(D-Phe)- 184 ntΨΛΨR (D-Asn)-(D-Thr)-Phg-F₂Pmp-Phg-185 IPΨΛΩR Ile-Pro-Phg-F₂Pmp-Nle- 186 QΘΣΛΘR Gln-Pip-Fpa-F₂Pmp-Pip- 187nAΣΛGR (D-Asn)-Ala-Fpa-F₂Pmp-Gly- 188 ntYΛAR(D-Asn)-(D-Thr)-Tyr-F₂Pmp-Ala- 189 cAΨΛvR (D-Glu)-Ala-Phg-F₂Pmp-(D-Val)-190 IvΨΛAR Ile-(D-Val)-Phg-F₂Pmp-Ala- 191 YtΨΛARTyr-(D-Thr)-Phg-F₂Pmp-Ala- 192 nΘΨΛIR (D-Asn)-Pip-Phg-F₂Pmp-Ile- 193ΘnWΛHR Pip-(D-Asn)-Trp-F₂Pmp-His- 194 YΘvΛIR Tyr-Pip-(D-Val)-F₂Pmp-Ile-195 nSAΛGR (D-Asn)-Ser-(D-Ala)-F₂Pmp-Gly- 196 tnvΛaR(D-Thr)-(D-Asn)-(D-Val)-F₂Pmp-(D-Ala)- 197 ntvΛtR(D-Asn)-(D-Thr)-(D-Val)-F₂Pmp-(D-Thr)- 198 SItΛYRSer-Ile-(D-Thr)-F₂Pmp-Tyr- 199 nΣnΛlR (D-Asn)-Fpa-(D-Asn)-F₂Pmp-(D-Leu)-200 YnnΛΩR Tyr-(D-Asn)-(D-Asn)-F₂Pmp-Nle- 201 nYnΛGR(D-Asn)-Tyr-(D-Asn)-F₂Pmp-Gly- 202 AWnΛAR Ala-Trp-(D-Asn)-F₂Pmp-Ala- 203vtHΛYR (D-Val)-(D-Thr)-His-F₂Pmp-Tyr- 204 PΨHΛΘR Pro-Phg-His-F₂Pmp-Pip-205 nΨHΛGR (D-Asn)-Phg-His-F₂Pmp-Gly- 206 PAHΛGR Pro-Ala-His-F₂Pmp-Gly-207 AYHΛIR Ala-Tyr-His-F₂Pmp-Ile- 208 nΘeΛYR(D-Asn)-Pip-(D-Glu)-F₂Pmp-Tyr- 209 vSSΛtR (D-Val)-Ser-Ser-F₂Pmp-(D-Thr)-210 aΞt′ ϑ Φ′YNK ((D-Ala)-Sar-(D-pThr)-Pp-Nal-Tyr-Gln)-Lys 211Tm(aΞt′ϑΦ′RA)Dap Tm((D-Ala)-Sar-(D-pThr)-Pp-Nal-Arg-Ala)-Dap 212Tm(aΞtϑΦ′RAa)Dap Tm((D-Ala)-Sar-(D-pThr)-Pp-Nal-Arg-Ala-(D- Ala))-Dap213 Tm(aΞtϑΦ′RAa)Dap Tm((D-Ala)-Sar-(D-Thr)-Pp-Nal-Arg-Ala-(D- Ala))-Dap214 Tm(aΞtaΦ′RAa)Dap Tm((D-Ala)-Sar-(D-Thr)-(D-Ala)-Nal-Arg-Ala-(D-Ala))-Dap * Fpa, Σ = L-4-fluorophenylalanine; Pip, Θ = L-homoproline;Nle, Ω = L-norleucine; Phg, Ψ = L-phenylglycine; F₂Pmp,Λ = L-4-(phosphonodifluoromethyl)phenylalanine; Dap= L-2,3-diaminopropionic acid; Nal, Φ′ = L-β-naphthylalanine; Pp,ϑ = L-pipecolic acid; Sar, Ξ = sarcosine; Tm = trimesic acid.

In some examples, the targeting moeity can by any of SEQ ID NO:168 toSEQ ID NO:214. In some examples, the targeting moiety can be a variantof any of SEQ ID NO:168 to SEQ ID NO:214.

The targeting moitiey and cell penetrating peptide moiety can overlap,that is residues that form the cell penetrating peptide moiety can alsobe part of the sequence that forms the targeting moiety, and vice aversa.

The therapeutic moiety can be attached to the cell penetrating peptidemoiety at the amino group, the carboxylate group, or the side chain ofany of the amino acids of the cell penetrating peptide moiety or cargomoiety (e.g., at the amino group, the carboxylate group, or the sidechain or any of X_(m), X_(n) or R¹). In some examples, the therapeuticmoiety can be attached to the detectable moiety.

In some examples, the therapeutic moiety can comprise a targeting moietythat can act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1,Grb2 SH2, CAL PDZ, and the like, or combinations thereof.

Ras is a protein that in humans is encoded by the RAS gene. The normalRas protein performs an essential function in normal tissue signaling,and the mutation of a Ras gene is implicated in the development of manycancers. Ras can act as a molecular on/off switch, once it is turned onRas recruits and activates proteins necessary for the propagation ofgrowth factor and other receptors' signal. Mutated forms of Ras havebeen implicated in various cancers, including lung cancer, colon cancer,pancreatic cancer, and various leukemias.

Protein-tyrosine phosphatase 1B (PTP1B) is a prototypical member of thePTP superfamily and plays numerous roles during eukaryotic cellsignaling. PTP1B is a negative regulator of the insulin signalingpathway, and is considered a promising potential therapeutic target, inparticular for the treatment of type II diabetes. PIP1B has also beenimplicated in the development of breast cancer.

Pin1 is an enzyme that binds to a subset of proteins and plays a role asa post phosphorylation control in regulating protein function. Pin1activity can regulate the outcome of proline-directed kinase signalingand consequently can regulate cell proliferation and cell survival.Deregulation of Pin1 can play a role in various diseases. Theup-regulation of Pin1 may be implicated in certain cancers, and thedown-regulation of Pin1 may be implicated in Alzheimer's disease.Inhibitors of Pin1 can have therapeutic implications for cancer andimmune disorders.

Grb2 is an adaptor protein involved in signal transduction and cellcommunication. The Grb2 protein contains one SH2 domain, which can bindtyrosine phosphorylated sequences. Grb2 is widely expressed and isessential for multiple cellular functions. Inhibition of Grb2 functioncan impair developmental processes and can block transformation andproliferation of various cell types.

It was recently reported that the activity of cystic fibrosis membraneconductance regulator (CFTR), a chloride ion channel protein mutated incystic fibrosis (CF) patients, is negatively regulated byCFTR-associated ligand (CAL) through its PDZ domain (CAL-PDZ) (Wolde, Met al. J. Biol. Chem. 2007, 282, 8099). Inhibition of the CFTR/CAL-PDZinteraction was shown to improve the activity of ΔPhe508-CFTR, the mostcommon form of CFTR mutation (Cheng, S H et al. Cell 1990, 63, 827;Kerem, B S et al. Science 1989, 245, 1073), by reducing itsproteasome-mediated degradation (Cushing, P R et al. Angew. Chem. Int.Ed. 2010, 49, 9907). Thus, disclosed herein is a method for treating asubject having cystic fibrosis by administering an effective amount of acompound or composition disclosed herein. The compound or compositionadministered to the subject can comprise a therapeutic moiety that cancomprise a targeting moiety that can act as an inhibitor against CALPDZ. Also, the dcompositions or compositions disclosed herein can beadministered with a molecule that corrects the CFTR function.

In some examples the targeting moiety can comprise E-T-G-E-F-L (SEQ IDNO:215) or LDPETGE (SEQ ID NO:216).

Linking Moiety

The disclosure provides for a compound according to Formula IV′:

wherein:

-   -   each Y is independently CH or N, provided no more than four Y        are N;    -   Z is OR_(a), hydrogen, halogen, carbocyclyl, herterocyclyl, or        an amino acid;    -   each R is independently an alkyl, alkenyl, alkynyl, carbocyclyl,        heterocyclyl, or an amino acid; and    -   R_(a) is independently H, C(O)alkyl, alkyl, alkenyl, alkynyl,        carbocyclic, or heterocyclyl.

In embodiments, the compound of Formula IV has a structure according toFormula IV′a:

In some embodiments, wherein Z is OH. In some embodiments, R isindependently aryl or hetereoaryl.

In some embodiments, the compound of Formula IV has a structureaccording to Formula IV′b

wherein Q at each instance is independently CH or N.

In some embodiments, the compound has the following structure

Methods of Making

The compounds described herein can be prepared using synthetictechniques known to one skilled in the art of organic synthesis orvariations thereon as appreciated by those skilled in the art. Thecompounds described herein can be prepared from readily availablestarting materials. Optimum reaction conditions can vary with theparticular reactants or solvents used, but such conditions can bedetermined by one skilled in the art.

Variations on the compounds described herein include the addition,subtraction, or movement of the various constituents as described foreach compound. Similarly, when one or more chiral centers are present ina molecule, the chirality of the molecule can be changed. Additionally,compound synthesis can involve the protection and deprotection ofvarious chemical groups. The use of protection and deprotection, and theselection of appropriate protecting groups can be determined by oneskilled in the art. The chemistry of protecting groups can be found, forexample, in Wuts and Greene, Protective Groups in Organic Synthesis, 4thEd., Wiley & Sons, 2006, which is incorporated herein by reference inits entirety.

The starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics(Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St.Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck(Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis(Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel,Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY),Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (AbbottPark, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim(Ingelheim, Germany), or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989). Othermaterials, such as the pharmaceutical carriers disclosed herein can beobtained from commercial sources.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

The disclosed compounds can be prepared by solid phase peptide synthesiswherein the amino acid α-N-terminal is protected by an acid or baseprotecting group. Such protecting groups should have the properties ofbeing stable to the conditions of peptide linkage formation while beingreadily removable without destruction of the growing peptide chain orracemization of any of the chiral centers contained therein. Suitableprotecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc),t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz),biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularlypreferred for the synthesis of the disclosed compounds. Other preferredside chain protecting groups are, for side chain amino groups likelysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc),nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, andadamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl,2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyland acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; forhistidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl;for tryptophan, formyl; for asparticacid and glutamic acid, benzyl andt-butyl and for cysteine, triphenylmethyl (trityl). In the solid phasepeptide synthesis method, the α-C-terminal amino acid is attached to asuitable solid support or resin. Suitable solid supports useful for theabove synthesis are those materials which are inert to the reagents andreaction conditions of the stepwise condensation-deprotection reactions,as well as being insoluble in the media used. Solid supports forsynthesis of α-C-terminal carboxy peptides is4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene) or4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif). The α-C-terminalamino acid is coupled to the resin by means ofN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC)or 0-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediatedcoupling for from about 1 to about 24 hours at a temperature of between10° C. and 50° C. in a solvent such as dichloromethane or DMF. When thesolid support is4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin,the Fmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the α-C-terminal amino acid as described above.One method for coupling to the deprotected 4(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin isO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. Thecoupling of successive protected amino acids can be carried out in anautomatic polypeptide synthesizer. In one example, the α-N-terminal inthe amino acids of the growing peptide chain are protected with Fmoc.The removal of the Fmoc protecting group from the α-N-terminal side ofthe growing peptide is accomplished by treatment with a secondary amine,preferably piperidine. Each protected amino acid is then introduced inabout 3-fold molar excess, and the coupling is preferably carried out inDMF. The coupling agent can beO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At the endof the solid phase synthesis, the polypeptide is removed from the resinand deprotected, either in successively or in a single operation.Removal of the polypeptide and deprotection can be accomplished in asingle operation by treating the resin-bound polypeptide with a cleavagereagent comprising thianisole, water, ethanedithiol and trifluoroaceticacid. In cases wherein the α-C-terminal of the polypeptide is analkylamide, the resin is cleaved by aminolysis with an alkylamine.Alternatively, the peptide can be removed by transesterification, e.g.with methanol, followed by aminolysis or by direct transamidation. Theprotected peptide can be purified at this point or taken to the nextstep directly. The removal of the side chain protecting groups can beaccomplished using the cleavage cocktail described above. The fullydeprotected peptide can be purified by a sequence of chromatographicsteps employing any or all of the following types: ion exchange on aweakly basic resin (acetate form); hydrophobic adsorption chromatographyon underivitized polystyrene-divinylbenzene (for example, AmberliteXAD); silica gel adsorption chromatography; ion exchange chromatographyon carboxymethylcellulose; partition chromatography, e.g. on SephadexG-25, LH-20 or countercurrent distribution; high performance liquidchromatography (HPLC), especially reverse-phase HPLC on octyl- oroctadecylsilyl-silica bonded phase column packing.

In a specific method disclosed herein are methods of making a bicyclicpeptide comprising, contacting a compound of Formula IV:

wherein Q¹ and Q² are, independent of one another, chosen from CH or N;with a solid supported peptide having from 8 to 20 amino acid residues,wherein at least two residues are cysteine residues; and cleaving thepeptide from the solid support. Examples of suitable solid supports arepolystyrene, polyacrylamide, polyethylene glycol supports. Rink, Wang,or Tentagel resins are suitable examples of solid supports that can beused. Cleaving the peptide from the solid supports can typically beaccomplished with mild acid or base.

Methods of Use

Also provided herein are methods of use of the compounds or compositionsdescribed herein. Also provided herein are methods for treating adisease or pathology in a subject in need thereof comprisingadministering to the subject an effective amount of any of the compoundsor compositions described herein.

Also provided herein are methods of treating, preventing, orameliorating cancer in a subject. The methods include administering to asubject an effective amount of one or more of the compounds orcompositions described herein, or a pharmaceutically acceptable saltthereof. The compounds and compositions described herein orpharmaceutically acceptable salts thereof are useful for treating cancerin humans, e.g., pediatric and geriatric populations, and in animals,e.g., veterinary applications. The disclosed methods can optionallyinclude identifying a patient who is or can be in need of treatment of acancer. Examples of cancer types treatable by the compounds andcompositions described herein include bladder cancer, brain cancer,breast cancer, colorectal cancer, cervical cancer, gastrointestinalcancer, genitourinary cancer, head and neck cancer, lung cancer, ovariancancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer,and testicular cancer. Further examples include cancer and/or tumors ofthe anus, bile duct, bone, bone marrow, bowel (including colon andrectum), eye, gall bladder, kidney, mouth, larynx, esophagus, stomach,testis, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord,thyroid, vagina, vulva, uterus, liver, muscle, blood cells (includinglymphocytes and other immune system cells). Further examples of cancerstreatable by the compounds and compositions described herein includecarcinomas, Karposi's sarcoma, melanoma, mesothelioma, soft tissuesarcoma, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic,acute myeloid, chronic lymphocytic, chronic myeloid, and other), andlymphoma (Hodgkin's and non-Hodgkin's), and multiple myeloma. Furtherexamples of cancers treatable by the disclosed compounds are p53cancers, e.g., by using compounds where the cargo moiety is SEQ ID NO.:156, e.g., SEQ ID NO.:166 and SEQ ID NO.:167.

The methods of treatment or prevention of cancer described herein canfurther include treatment with one or more additional agents (e.g., ananti-cancer agent or ionizing radiation). The one or more additionalagents and the compounds and compositions or pharmaceutically acceptablesalts thereof as described herein can be administered in any order,including simultaneous administration, as well as temporally spacedorder of up to several days apart. The methods can also include morethan a single administration of the one or more additional agents and/orthe compounds and compositions or pharmaceutically acceptable saltsthereof as described herein. The administration of the one or moreadditional agents and the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein can be by the same ordifferent routes. When treating with one or more additional agents, thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein can be combined into a pharmaceutical compositionthat includes the one or more additional agents.

For example, the compounds or compositions or pharmaceuticallyacceptable salts thereof as described herein can be combined into apharmaceutical composition with an additional anti-cancer agent.

The compounds disclosed herein can also be used alone or in combinationwith anticancer or antiviral agents, such as ganciclovir, azidothymidine(AZT), lamivudine (3TC), etc., to treat patients infected with a virusthat can cause cellular transformation and/or to treat patients having atumor or cancer that is associated with the presence of viral genome inthe cells. The compounds disclosed herein can also be used incombination with viral based treatments of oncologic disease.

Also described herein are methods of killing a tumor cell in a subject.The method includes contacting the tumor cell with an effective amountof a compound or composition as described herein, and optionallyincludes the step of irradiating the tumor cell with an effective amountof ionizing radiation. Additionally, methods of radiotherapy of tumorsare provided herein. The methods include contacting the tumor cell withan effective amount of a compound or composition as described herein,and irradiating the tumor with an effective amount of ionizingradiation. As used herein, the term ionizing radiation refers toradiation comprising particles or photons that have sufficient energy orcan produce sufficient energy via nuclear interactions to produceionization. An example of ionizing radiation is x-radiation. Aneffective amount of ionizing radiation refers to a dose of ionizingradiation that produces an increase in cell damage or death whenadministered in combination with the compounds described herein. Theionizing radiation can be delivered according to methods as known in theart, including administering radiolabeled antibodies and radioisotopes.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. As used herein the term treatingor treatment includes prevention; delay in onset; diminution,eradication, or delay in exacerbation of signs or symptoms after onset;and prevention of relapse. For prophylactic use, a therapeuticallyeffective amount of the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein are administered to asubject prior to onset (e.g., before obvious signs of cancer), duringearly onset (e.g., upon initial signs and symptoms of cancer), or afteran established development of cancer. Prophylactic administration canoccur for several days to years prior to the manifestation of symptomsof an infection. Prophylactic administration can be used, for example,in the chemopreventative treatment of subjects presenting precancerouslesions, those diagnosed with early stage malignancies, and forsubgroups with susceptibilities (e.g., family, racial, and/oroccupational) to particular cancers. Therapeutic treatment involvesadministering to a subject a therapeutically effective amount of thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein after cancer is diagnosed.

In some examples of the methods of treating of treating, preventing, orameliorating cancer or a tumor in a subject, the compound or compositionadministered to the subject can comprise a therapeutic moiety that cancomprise a targeting moiety that can act as an inhibitor against Ras(e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, or combinations thereof.

The disclosed subject matter also concerns methods for treating asubject having a metabolic disorder or condition. In one embodiment, aneffective amount of one or more compounds or compositions disclosedherein is administered to a subject having a metabolic disorder and whois in need of treatment thereof. In some examples, the metabolicdisorder can comprise type II diabetes. In some examples of the methodsof treating of treating, preventing, or ameliorating the metabolicdisorder in a subject, the compound or composition administered to thesubject can comprise a therapeutic moiety that can comprise a targetingmoiety that can act as an inhibitor against PTP1B. In one particularexample of this method the subject is obese and the method comprisestreating the subject for obesity by administering a composition asdisclosed herein.

The disclosed subject matter also concerns methods for treating asubject having an immune disorder or condition. In one embodiment, aneffective amount of one or more compounds or compositions disclosedherein is administered to a subject having an immune disorder and who isin need of treatment thereof. In some examples of the methods oftreating of treating, preventing, or ameliorating the immune disorder ina subject, the compound or composition administered to the subject cancomprise a therapeutic moiety that can comprise a targeting moiety thatcan act as an inhibitor against Pin1.

The disclosed subject matter also concerns methods for treating asubject having cystic fibrosis. In one embodiment, an effective amountof one or more compounds or compositions disclosed herein isadministered to a subject having cystic fibrosis and who is in need oftreatment thereof. In some examples of the methods of treating thecystic fibrosis in a subject, the compound or composition administeredto the subject can comprise a therapeutic moiety that can comprise atargeting moiety that can act as an inhibitor against CAL PDZ.

Compositions, Formulations and Methods of Administration

In vivo application of the disclosed compounds, and compositionscontaining them, can be accomplished by any suitable method andtechnique presently or prospectively known to those skilled in the art.For example, the disclosed compounds can be formulated in aphysiologically- or pharmaceutically-acceptable form and administered byany suitable route known in the art including, for example, oral, nasal,rectal, topical, and parenteral routes of administration. As usedherein, the term parenteral includes subcutaneous, intradermal,intravenous, intramuscular, intraperitoneal, and intrasternaladministration, such as by injection. Administration of the disclosedcompounds or compositions can be a single administration, or atcontinuous or distinct intervals as can be readily determined by aperson skilled in the art.

The compounds disclosed herein, and compositions comprising them, canalso be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time. The compounds can also be administered in theirsalt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to knownmethods for preparing pharmaceutically acceptable compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin (1995)describes formulations that can be used in connection with the disclosedmethods. In general, the compounds disclosed herein can be formulatedsuch that an effective amount of the compound is combined with asuitable carrier in order to facilitate effective administration of thecompound. The compositions used can also be in a variety of forms. Theseinclude, for example, solid, semi-solid, and liquid dosage forms, suchas tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically-acceptable carriers and diluents which areknown to those skilled in the art. Examples of carriers or diluents foruse with the compounds include ethanol, dimethyl sulfoxide, glycerol,alumina, starch, saline, and equivalent carriers and diluents. Toprovide for the administration of such dosages for the desiredtherapeutic treatment, compositions disclosed herein can advantageouslycomprise between about 0.1% and 100% by weight of the total of one ormore of the subject compounds based on the weight of the totalcomposition including carrier or diluent.

Formulations suitable for administration include, for example, aqueoussterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions, which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions disclosed herein can include other agents conventional inthe art having regard to the type of formulation in question.

Compounds disclosed herein, and compositions comprising them, can bedelivered to a cell either through direct contact with the cell or via acarrier means. Carrier means for delivering compounds and compositionsto cells are known in the art and include, for example, encapsulatingthe composition in a liposome moiety. Another means for delivery ofcompounds and compositions disclosed herein to a cell comprisesattaching the compounds to a protein or nucleic acid that is targetedfor delivery to the target cell. U.S. Pat. No. 6,960,648 and U.S.Application Publication Nos. 20030032594 and 20020120100 disclose aminoacid sequences that can be coupled to another composition and thatallows the composition to be translocated across biological membranes.U.S. Application Publication No. 20020035243 also describes compositionsfor transporting biological moieties across cell membranes forintracellular delivery. Compounds can also be incorporated intopolymers, examples of which include poly (D-L lactide-co-glycolide)polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL);chondroitin; chitin; and chitosan.

For the treatment of oncological disorders, the compounds disclosedherein can be administered to a patient in need of treatment incombination with other antitumor or anticancer substances and/or withradiation and/or photodynamic therapy and/or with surgical treatment toremove a tumor. These other substances or treatments can be given at thesame as or at different times from the compounds disclosed herein. Forexample, the compounds disclosed herein can be used in combination withmitotic inhibitors such as taxol or vinblastine, alkylating agents suchas cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracilor hydroxyurea, DNA intercalators such as adriamycin or bleomycin,topoisomerase inhibitors such as etoposide or camptothecin,antiangiogenic agents such as angiostatin, antiestrogens such astamoxifen, and/or other anti-cancer drugs or antibodies, such as, forexample, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN(Genentech, Inc.), respectively, or an immunotherapeutic such asipilimumab and bortezomib.

In certain examples, compounds and compositions disclosed herein can belocally administered at one or more anatomical sites, such as sites ofunwanted cell growth (such as a tumor site or benign skin growth, e.g.,injected or topically applied to the tumor or skin growth), optionallyin combination with a pharmaceutically acceptable carrier such as aninert diluent. Compounds and compositions disclosed herein can besystemically administered, such as intravenously or orally, optionallyin combination with a pharmaceutically acceptable carrier such as aninert diluent, or an assimilable edible carrier for oral delivery. Theycan be enclosed in hard or soft shell gelatin capsules, can becompressed into tablets, or can be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, the activecompound can be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, aerosol sprays, and the like.

The disclosed compositions are bioavailable and can be delivered orally.Oral compositions can be tablets, troches, pills, capsules, and thelike, and can also contain the following: binders such as gumtragacanth, acacia, corn starch or gelatin; excipients such as dicalciumphosphate; a disintegrating agent such as corn starch, potato starch,alginic acid and the like; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, fructose, lactose or aspartame or aflavoring agent such as peppermint, oil of wintergreen, or cherryflavoring can be added. When the unit dosage form is a capsule, it cancontain, in addition to materials of the above type, a liquid carrier,such as a vegetable oil or a polyethylene glycol. Various othermaterials can be present as coatings or to otherwise modify the physicalform of the solid unit dosage form. For instance, tablets, pills, orcapsules can be coated with gelatin, wax, shellac, or sugar and thelike. A syrup or elixir can contain the active compound, sucrose orfructose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, the active compound can be incorporated intosustained-release preparations and devices.

Compounds and compositions disclosed herein, including pharmaceuticallyacceptable salts or prodrugs thereof, can be administered intravenously,intramuscularly, or intraperitoneally by infusion or injection.Solutions of the active agent or its salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compoundand/or agent disclosed herein in the required amount in the appropriatesolvent with various other ingredients enumerated above, as required,followed by filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, compounds and agents disclosed herein can beapplied in as a liquid or solid. However, it will generally be desirableto administer them topically to the skin as compositions, in combinationwith a dermatologically acceptable carrier, which can be a solid or aliquid. Compounds and agents and compositions disclosed herein can beapplied topically to a subject's skin to reduce the size (and caninclude complete removal) of malignant or benign growths, or to treat aninfection site. Compounds and agents disclosed herein can be applieddirectly to the growth or infection site. Preferably, the compounds andagents are applied to the growth or infection site in a formulation suchas an ointment, cream, lotion, solution, tincture, or the like.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Useful dosages of the compounds and agents and pharmaceuticalcompositions disclosed herein can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art.

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptoms ordisorder are affected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

Also disclosed are pharmaceutical compositions that comprise a compounddisclosed herein in combination with a pharmaceutically acceptablecarrier. Pharmaceutical compositions adapted for oral, topical orparenteral administration, comprising an amount of a compound constitutea preferred aspect. The dose administered to a patient, particularly ahuman, should be sufficient to achieve a therapeutic response in thepatient over a reasonable time frame, without lethal toxicity, andpreferably causing no more than an acceptable level of side effects ormorbidity. One skilled in the art will recognize that dosage will dependupon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

Also disclosed are kits that comprise a compound disclosed herein in oneor more containers. The disclosed kits can optionally includepharmaceutically acceptable carriers and/or diluents. In one embodiment,a kit includes one or more other components, adjuncts, or adjuvants asdescribed herein. In another embodiment, a kit includes one or moreanti-cancer agents, such as those agents described herein. In oneembodiment, a kit includes instructions or packaging materials thatdescribe how to administer a compound or composition of the kit.Containers of the kit can be of any suitable material, e.g., glass,plastic, metal, etc., and of any suitable size, shape, or configuration.In one embodiment, a compound and/or agent disclosed herein is providedin the kit as a solid, such as a tablet, pill, or powder form. Inanother embodiment, a compound and/or agent disclosed herein is providedin the kit as a liquid or solution. In one embodiment, the kit comprisesan ampoule or syringe containing a compound and/or agent disclosedherein in liquid or solution form.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric.

Example 1. Synthesis of Bicyclizatoin Scaffold

Reagents for peptide synthesis were purchased from Chem-Impex (WoodDale, IL), NovaBiochem (La Jolla, CA), or Anaspec (San Jose, CA). Rinkamide resin LS (100-200 mesh, 0.2 mmol/g) was purchased from AdvancedChemTech. Cell culture media, fetal bovine serum,penicillin-streptomycin, 0.25% trypsin-EDTA, and DPBS were purchasedfrom Invitrogen (Carlsbad, CA). Methyl 3,5-dimethylbenzoiate,N-bromosuccinimide, diethyl phosphite, 2,2′-dipyridyl disulfide, andother organic reagents/solvents were purchased from Sigma-Aldrich (St.Louis, MO). Anti-GST-Tb and streptavidin-d2 were purchased from Cisbio(Bedford, MA). The NF-κB reporter (Luc)-HEK293 cell line and One-Step™luciferase assay system were purchased from BPS Bioscience (San Diego,CA).

Synthesis of Methyl 3,5-Bis(bromomethyl)benzoate

The overall synthetic plan for the bicyclization scaffold is shown inScheme 2. To a 50-mL round-bottom flask charged with methyl3,5-dimethylbenzoate (2.0 g, 12.2 mmol) in carbon tetrachloride (20 mL,sparged with nitrogen) was added N-bromosuccinimide (4.25 g, 23.9 mmol)and benzoyl peroxide (˜60 mg) as an initiator. The reaction was refluxedfor 3 h under nitrogen atmosphere. The reaction mixture was cooled, andfiltered. The filtrate was washed with water (20 mL), dried with MgSO₄,and concentrated in vacuo. The crude product was recrystallized inpetroleum ether to yield the title compound (1.2 g, 3.8 mmol) in 30%yield. ¹H NMR (300 MHz, CDCl₃): δ 7.99 (s, 2H), 7.62 (s, 1H), 4.50 (s,4H), 3.94 (s, 3H).

Synthesis of Methyl 3,5-bis((acetylthio)methyl)benzoate

To a 50-mL round-bottom flask charged with crude methyl3,5-bis(bromomethyl)benzoate (1.0 g, 3.1 mmol) from above was addedacetone (20 mL) and potassium thioacetate (0.86 g, 7.52 mmol). Thereaction was refluxed for 3 h under nitrogen atmosphere and allowed tocool. 20 mL water was added to quench the reaction and the mixture wasextracted with ethyl acetate. The combined organic layer was dried withMgSO₄, concentrated, and purified by silica gel chromatography to afford0.72 g of an orange-brown solid (74.2% yield). ¹H NMR (300 MHz, CDCl₃):δ 7.83 (s, 2H), 7.41 (s, 1H), 4.12 (s, 4H), 3.90 (s, 3H), 2.36 (s, 6H).

Synthesis of 3,5-bis((pyridin-2-yldisulfanyl)methyl)benzoic acid

A 50-mL round-bottom flask under nitrogen atmosphere was charged withmethyl 3,5-bis((acetylthio)methyl)benzoate (0.5 g, 1.6 mmol) dissolvedin MeOH (15 mL). A solution of NaOH (832 mg, 20.8 mmol) in H₂O (3 mL)was added and the reaction was allowed to react overnight at roomtemperature. The reaction solution was acidified with AcOH (2.38 mL,41.6 mmol) and 2,2′-dipyridyl disulfide (1.41 g, 6.4 mmol) was added.The reaction mixture was filtered to remove the orange precipitateformed and allowed to stir for 1 h at room temperature. After thereaction was complete, the methanol was removed by evaporation in vacuoand the residue was quickly loaded onto a silica gel column. The columnwas first eluted with 20% to 50% EtOAc in hexanes to remove any lowpolarity species, after which the desired product was eluted with 1:1(v/v) EtOAc in hexanes containing 1% AcOH. Evaporation of the solventsgave a brown solid (222 mg, 32% yield over two steps). ¹H NMR (400 MHz,CDCl₃): δ 8.43 (m, 2H), 7.83 (s, 2H), 7.53-7.43 (m, 5H), 7.04 (m, 2H),3.97 (s, 4H). HRMS (ESI+): calcd for C₁₉H₁₇N₂O₂S₄ (M+H⁺): 433.0173;Found: 433.0176.

Example 2. Synthesis of Bicyclic Peptides

Peptide Preparation and Characterization.

Peptides were synthesized on Rink amide resin LS (0.2 mmol/g) usingstandard Fmoc chemistry. A typical coupling reaction contained 5 equivof Fmoc-amino acid, 5 equiv of2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), and 10 equiv of diisopropylethylamine(DIPEA) and was allowed to proceed for 45 min with gentle mixing. Thepeptides were deprotected and released from the resin by treatment with90:2.5:2.5:2.5:2.5 (v/v)TFA/1,2-ethanedithiol/water/phenol/triisopropylsilane (TIPS) for 2 h.The peptides were triturated with cold ethyl ether (3×) and purified byreversed-phase HPLC equipped with a C₁₈ column. Peptide labeling withfluorescein isothiocyanate (FITC) was performed by dissolving thepurified peptides (˜1 mg each) in 300 μL of 1:1:1 DMSO/DMF/150 mM sodiumbicarbonate (pH 8.5) and mixing with 10 μL of FITC in DMSO (100 mg/mL).After 20 min at room temperature, the reaction mixture was purifiedagain by reversed-phase HPLC to isolate the FITC-labeled peptide.

To synthesize the disulfide-mediated bicyclic peptides, thecorresponding linear peptides containing two Acm-protected Cys residueswere first prepared using standard Fmoc/HATU chemistry. The Acm groupswere removed by treating the resin with 2 M mercury(II) acetate in DMFovernight. The resin was then incubated in 20% (3-mercaptoethanol in DMFfor 2 h (twice) to release the free thiol. After exhaustive washing withDMF to remove all of the reducing agents, the resin was incubatedovernight with 1 equiv. of3,5-bis((pyridin-2-yldisulfanyl)methyl)benzoic acid in methanolcontaining 1% (v/v) acetic acid. The reaction progress was monitored byremoving a small portion of the resin and analyzing thedeprotected/released peptide product by MALDI-TOF MS. Peptidedeprotection and release were achieved by treating the resin with85:10:2.5:2.5 (v/v) TFA/DCM/water/TIPS for 2 h, followed by ethertrituration and HPLC purification as described above (FIG. 5A). All ofthe final peptides used in this work had ≥95% purity as judged byanalytical HPLC (FIG. 5B and FIG. 7 ). The authenticity of the peptideswas confirmed by MALDI-TOF MS analysis. To further characterize thebiologically active peptide 4, the peptide was be dissolved in H₂O/D₂O(9:1, 500 μL; final sample concentration 2 mM). NMR spectra wererecorded on a Bruker Ascend 700 MHz spectrometer at 298 K.

Example 3. Cell-Based Assays

Cell Culture.

HeLa cells were maintained in media consisting of DMEM, 10% fetal bovineserum (FBS) and 1% penicillin/streptomycin. The NF-κB reporter(Luc)-HEK293 cells were maintained in media consisting of DMEM, 10% FBS,1% penicillin/streptomycin, and 100 μg/ml of hygromycin B. Cells werecultured in a humidified incubator at 37° C. in the presence of 5% CO₂.

Protein Expression and Purification.

Escherichia coli BL21(DE3) cells were transformed with apGEX4T3-GST-NEMO(1-196) plasmid and grown at 37° C. in Luria brothsupplemented with 0.05 mg/mL ampicillin to an OD₆₀₀ of 0.4. Expressionwas induced by the addition of isopropyl (3-D-1-thiogalactopyranoside(150 μM final concentration). After five hours at 30° C., the cells wereharvested by centrifugation. The cell pellet was suspended in 40 mL oflysis buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5 mM MgCl₂, 5 mMβ-mercaptoethanol, 0.1% Triton-X-100, pH 8.0), 100 μg/mL lysozyme, 100μl DNAse I (New England BioLabs), and 100 μl of Halt Protease Inhibitorcocktail (EDTA-free) (Thermo Scientific). This mixture was stirred at 4°C. for 30 min and briefly sonicated (2×10 s pulses). The crude lysatewas centrifuged to yield a clear supernatant, which was directly loadedonto a glutathione-Sepharose 4B column (GE Healthcare). The boundprotein was eluted from the column with 10 mM Glutathione in 50 mMTris-HCl (pH 8.0) (40 mL), concentrated to 0.5 mL with the use of AmiconUltra-15 centrifugal filter units (MWCO 10 kDa), and dialyzed againstPBS (2.67 mM potassium chloride, 1.47 mM potassium phosphate monobasic,137 mM sodium chloride, and 8.06 mM sodium phosphate dibasic). Togenerate NEMO without the GST tag, the protein was treated with thrombin(GE Healthcare) for 16 h at 4° C. prior to concentration. Proteinconcentration was determined using Bradford assay with bovine serumalbumin as the standard. The protein was quickly frozen and stored at−80° C.

An engineered prokaryotic expression plasmid pJCC04a,1 which encodes afusion protein containing an N-terminal six-histidine tag, thioredoxin,a TEV protease cleavage site, and the K703R/K704R mutant form of IKKPC-terminal fragment (amino acids 701-745)[His-thx-IKKβ_(KK/RR)(701-745)], was kindly provided by Dr. MariaPellegrini (Dartmouth College). His-thx-IKKβ_(KK/RR)(701-745) wassimilarly expressed in E. coli BL21 (DE3) cells and purified by affinitychromatography using a HisTrap FF column (GE Healthcare). The fusionprotein was eluted with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 250 mMImadizole, 2 mM β-mercaptoethanol and treated with TEV protease (150units for 1 mg of fusion protein) for 16 h at 4° C. to remove thethioredoxin (thx). The resulting protease digestion mixture wasre-loaded onto the HisTrap column. The flow-through fraction wascollected and concentrated to ˜2 mg/mL using Amicon Ultra-15 centrifugalfilter units (MWCO 10 kDa). The IKKβ_(KK/RR)(701-745) protein wasbiotinylated by treatment with a 10-fold molar excess of biotin-NHS at4° C. overnight. The biotinylated IKKβ_(KK/RR)(701-745) was purified byreversed-phase HPLC equipped with a C₁₈ column and stored frozen at −80°C.

Flow Cytometry.

HeLa cells were cultured in six-well plates (5×10⁵ cells per well) for24 h. On the day of experiment, the cells were incubated with 5 μMFITC-labeled peptide in clear DMEM with 1% FBS at 37° C. for 2 h. Thecells were washed with DPBS, detached from plate with 0.25% trypsin,diluted into clear DMEM containing 10% FBS, pelleted at 250 g for 5 min,washed once with DPBS and resuspended in DPBS containing 1% bovine serumalbumin, and analyzed on a BD LSR II flow cytometer. Data were analyzedwith Flowjo software (Tree Star).

Serum Stability Test.

The stability tests were carried by modifying a previously reportedprocedure.² Diluted human serum (25%) was centrifuged at 15,000 rpm for10 min, and the supernatant was collected. A peptide stock solution wasdiluted into the supernatant to a final concentration of 5 μM andincubated at 37° C. At various time points (0-20 h), 200-μL aliquotswere withdrawn and mixed with 50 μL of 15% trichloroacetic acid and 200μL of acetonitrile, and the mixture was incubated at 4° C. overnight.The final mixture was centrifuged at 15,000 rpm for 10 min in amicrocentrifuge, and the supernatant was analyzed by reversed-phase HPLCequipped with an analytical C₁₈ column (Waters). The amount of remainingpeptide (%, relative to the time zero control) was determined byintegrating the area underneath the peptide peak (monitored at 214 nm).

HTRF Assay.

Recombinant GST-NEMO (30 nM), biotin-IKKβ_(KK/RR)(701-745) (50 nM),streptavidin labeled with d2 acceptor (2.5 μg/mL), anti-GST monoclonalantibody labeled with Tb donor (2.5 μg/mL), and varying concentrationsof peptide (0-100 μM) were mixed in PBS containing 5 mM TCEP and 0.01%Triton X-100 (total volume 20 μL) in a 384-well plate. The plate wasincubated for 2 h at room temperature. The HTRF signals were measured ona Tecan infinite M1000 Pro microplate reader and plotted as a functionof the peptide concentration. The data was analyzed using GraphPad Prism6.0 and IC50 values were obtained by fitting the data to thedose-response inhibition curves.

NF-κB Luciferase Assay.

NF-κB reporter (Luc)-HEK293 cells were seeded in 96-well microplate in45 μL of assay medium (DMEM, 10% FBS, and 1% penicillin/streptomycin,˜1500 cells per well) and cultured overnight. Five μL of NEMO inhibitorin assay medium was added to cells and the cells were incubated for 2 h.Recombinant TNFα³ in 5 μL of assay medium was added to the wells at thefinal concentration of 5 ng/mL. After 4 h of incubation, 55 μL ofONE-Step luciferase assay reagent was added to each well. Luminescencewas measured after 10 min of incubation using a Tecan Infinite M1000 Promicroplate reader. Luciferase activities of TNFα unstimulated andstimulated cells were recorded as AU⁻ and AU⁺, respectively. Luciferaseactivities of TNFα stimulated cells after incubating with differentconcentrations of NEMO inhibitors were recorded as AU^(pep). Theinhibition of NF-κB signaling activation is calculated by the percentageof luciferase activity induction based on the equation:Inhibition of TNFα Activation (%)=(AU ^(pep)-AU ⁻)/(AU ⁺ −AU ⁻)×100%

Results.

To test the validity of the reversible bicyclization strategy, two modelpeptides comprising the CPP motif (RRRRΦF (SEQ ID NO.:68) or FΦRRRR (SEQID NO.:69) and a mock cargo motif (SASAS ((SEQ ID NO.:156)) fused to itsN- or C-terminus (Table 6, peptides 2 and 3, FIG. 3 for detailedstructures) were designed. Two cysteine residues were also incorporatedinto the sequences for later cyclization, one at the junction betweenthe CPP and cargo motifs and one at the C-terminus. The linear peptideswere synthesized by standard Fmoc solid-phase peptide synthesis (SPPS)chemistry on Rink amide resin (Scheme 1). The acetamidomethyl (Acm)groups on the two cysteine side chains were selectively removed bytreatment with Hg(OAc)₂ and the exposed free thiols were then reactedon-resin with 3,5-bis((pyridin-2-yldisulfanyl)methyl)benzoic acid, whichwas readily prepared from commercially available starting materials(Scheme 2). Formation of two disulfide bonds between the cysteine sidechains and the 3,5-bis(mercaptomethyl)benzoic acid (BMB) scaffoldresulted in cyclization of the peptide. Next, the N-terminal Fmoc groupwas removed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and the peptidewas bicyclized by forming a lactam between the carboxyl group of BMB andthe N-terminal amine (Scheme 1). BMB is ideally suited as the scaffold,because its structural symmetry ensures that a single bicyclic productis formed following the disulfide exchange reactions. Additionally, therigidity of the scaffold prevents the formation of any intramoleculardisulfide bond, simplifying both the synthesis of the scaffold and itsreaction with the cysteine-containing peptides.

To monitor their cellular uptake, peptides 2 and 3 were labeled withfluorescein isothiocyanate (FITC) on the side chain of a C-terminallysine. Flow cytometry analysis of HeLa cells treated with 5 μM peptidescFΦR₄ (SEQ ID NO.:72), 2 and 3 for 2 h showed mean fluorescenceintensity (MFI) values of 3020, 5180, and 4100, respectively (FIG. 1A).Thus, bicyclic peptides 2 and 3 entered HeLa cells with 72% and 36%higher efficiencies, respectively, than cFΦR₄ (SEQ ID NO.:72).

The reversible bicyclization strategy was applied to generate acell-permeable, biologically active peptidyl inhibitor against theNEMO-IKK interaction. Despite of its in vivo efficacy, the linearAntp-NBD peptide has poor pharmacokinetics, due to rapid proteolyticdegradation in serum (t_(1/2)˜15 min). Conversion of Antp-NBD into aconformationally constrained bicyclic structure was envisioned tosubstantially increase its proteolytic stability. The CPP motif RRRRΦF(SEQ ID NO.:68) was fused to the N-terminus of NBD and the N- andC-terminal threonine residues were replaced with two cysteines (Table 6,peptide 4, FIG. 4 for detailed structure ((SEQ ID NO.:216). The peptidefusion was bicyclized around the BMB scaffold via two disulfide bonds asdescribed above, to give bicyclic peptide 4 as the predominant product(FIG. 5A). As a control, peptide 5 (FIG. 4 for detailed structure) wasalso prepared, which is structurally similar to peptide 4 but containstwo Ala residues in place of the two Trp residues. It was previouslyshown that replacement of the Trp residues with alanine largelyabolished NEMO binding (M. J. May, et al., Science 2000, 289, 1550).

Peptides 4 and 5 were labeled with FITC at the side chain of a lysineadded to their C-termini and their cellular entry was assessed by flowcytometry. Both peptides entered HeLa cells efficiently, exhibiting MFIvalues that were 3- and 2-fold higher than that of cFΦR₄, respectively(FIG. 1A). The NEMO-binding affinity of peptides 4 and 5 was determinedusing a homogenous time-resolved fluorescence (HTRF) assay (M. Rushe, etal., Structure 2008, 16, 798; Y. Gotoh, et al., Anal. Biochem. 2010,405, 19). Briefly, in the presence of an anti-glutathione-S-transferase(GST) antibody labeled with a fluorescence donor (Tb) and streptavidinlabeled with a fluorescence acceptor (d2), binding of GST-NEMO to abiotinylated IKKP fragment (amino acids 701-745) (B. Gao, et al.,Biochemistry 2014, 53, 677) results in a resonance energy transfer.Addition of a NEMO inhibitor blocks the NEMO-IKKβ interaction andreduces the HTRF signal. In the presence of 5 mMtris(carboxylethyl)phosphine (TCEP), which is expected to completelyreduce the disulfide bonds in peptides 4 and 5, peptide 4 inhibited theNEMO-IKKβ interaction in a concentration-dependent manner, with ahalf-maximal inhibitory concentration (IC₅₀) value of 3.5±0.2 μM (FIG.1B). Under the same conditions, Antp-NBD showed an IC₅₀ value of −50 μM,in agreement with the previously reported binding affinity (M. Rushe, etal., Structure 2008, 16, 798; Y. Gotoh, et al., Anal. Biochem. 2010,405, 19). As expected, up to 100 μM peptide 5 caused only minorinhibition of the interaction. Since substitution of the two cysteineresidues for threonine did not significantly change the NEMO bindingaffinity (FIG. 6 ), the enhanced NEMO binding of peptide 4 relative toAntp-NBD is likely caused by additional interactions between thephenylalanine of the CPP motif (RRRRΦF (SEQ ID NO.:68)) and the NEMOprotein surface. IKKβ contains a phenylalanine at the same position(Phe-734). The crystal structure of the NEMO-IKKβ complex shows that theside chain of Phe-734 inserts into a hydrophobic pocket on the NEMOsurface (Id.). Thus, the phenylalanine in peptide 4 likely plays dualroles of cellular entry and NEMO binding.

The ability of the bicyclic peptides to modulate the NEMO-IKKinteraction inside the cell was assessed by monitoring the TNFα-inducedactivation of NF-κB. HEK293 cells transfected with a luciferase reportergene under the control of NF-κB were first treated with varyingconcentrations of a peptide for 2 h and then TNFα (M. J. May, et al.,Science 2000, 289, 1550; A. Gaurnier-Hausser, et al., Clin. Cancer Res.2011, 17, 4661). In the absence of any inhibitory peptide, treatmentwith 5 ng/mL TNFα increased the luciferase activity from a basal levelof 177 arbitrary units (AU) to 715 AU. Peptide 4 reduced theTNFα-induced luciferase activity in a dose-dependent manner, with anIC₅₀ value of ˜20 μl (FIG. 1C). In contrast, the control peptide 5 hadno significant effect on NF-κB signaling at 20 μM and resulted in ˜10%inhibition at the highest concentration tested (40 μM). Consistent withthe earlier report (M. J. May, et al., Science 2000, 289, 1550),Antp-NBD (peptide 1) also caused concentration-dependent inhibition, butshowed an IC₅₀ value of 140 μM. The higher potency of bicyclic peptide 4relative to Antp-NBD in the cellular assay is likely the results of bothimproved cellular entry efficiency (FIG. 1A) and greater NEMO-bindingaffinity (FIG. 1B). In vitro treatment of bicyclic peptide 4 with 5 mMglutathione for 2 h completely reduced the disulfide bonds (FIG. 5B),suggesting that peptides 2-5 should undergo complete reduction uponcytosolic entry.

Finally, the proteolytic stability of peptide 4 and Antp-NBD was testedby incubating the peptides in human serum for varying lengths of timeand the remaining amounts of intact peptides were quantitated byanalytical HPLC. For comparison, a control peptide (Table 6, peptide 6)was synthesized, which has the same sequence as peptide 4 but only itsCPP motif was cyclized. In agreement with the previous reports (E. Jimi,et al., Nat. Med. 2004, 10, 617; S. Dai, et al., J. Biol. Chem. 2004,279, 37219; W. Shibata, et al., J. Immunol. 2007, 179, 2681; S. H. Dave,et al., J. Immunol. 2007, 179, 7852; A. Gaurnier-Hausser, et al., Clin.Cancer Res. 2011, 17, 4661; J. M. Peterson, et al., Mol. Med. 2011, 17,508; D. A. Delfin, et al., J. Transl. Med. 2011, 9, 68; D. P. Reay, etal., Neurobiol. Dis. 2011, 43, 598; J. N. Kornegay, et al., Skelet.Muscle 2014, 4, 18; G. Habineza Ndikuyeze, et al., PLoS One, 2014, 9,e95404), Antp-NBD was rapidly degraded by human serum, with a half-lifeof ˜15 min (FIG. 1D). In contrast, bicyclic peptide 4 showed a half-lifeof ˜10 h, and 23% of the peptide remained intact after 20 h ofincubation at 37° C. The monocyclic control peptide 6 was also rapidlydegraded (with a half-life of ˜30 min), likely due to proteolysis of thelinear NBD sequence. It was previously shown that linear peptidyl cargosattached to the Gln side chain of cFΦR₄ were rapidly degraded in humanserum (Z. Qian, et al., Angew. Chem. Int. Ed. 2015, 54, 5874; Angew.Chem. 2015, 127, 5972).

In conclusion, a simple method has been developed to efficiently deliverpeptidyl ligands into mammalian cells, by fusing the peptide with ashort CPP motif and reversibly cyclizing the fusion peptide throughdisulfide bonds. The resulting bicyclic peptide has greatly enhancedcellular uptake as well as proteolytic stability. This strategy shouldbe applicable to delivering any linear peptides.

Example 4. Synthesis of Bicyclic Peptide that Releases Peptidyl Cargofrom the Cyclic Cell-Penetrating Peptide in the Cytosol

As shown below in Scheme 2, the desired cyclic CPP was first synthesizedby standard solid-phase peptide synthesis using the Fmoc/HATU chemistryand anchored to the support through a Lys(Mtt) linker. While still onresin, the Mtt group is removed with 2% TFA and the exposed Lys sidechain is coupled to the bis(mercaptomethyl)benzoic acid scaffold byusing HATU. The cyclic CPP is then cleaved off the resin and deprotectedby TFA. The free thiols are protected/activated by reacting the peptidein solution (pH 5) with dithiodipyridine to generate the CPP-scaffold.Finally, the desired CPP-peptide cargo conjugate is prepared by simplymixing the CPP-scaffold and a thiol-containing peptide in an aqueousbuffer at pH 8.

Various bicyclic peptides synthesized according to Scheme 2.Specifically, cyclic CPP12 (FfΦRrRr) was conjugated to a peptidylinhibitor against Keap1-Nrf2 (FIG. 10A), a peptidyl inhibitor againstPin1 (FIG. 11 ), peptidyl inhibitor against the CAL PDZ-CFTR (PDZ)interaction (FIG. 12A), and a peptidyl inhibitor against the MDM2-p53interaction (PMI) (FIG. 13A). Fluorescent labels were attached usingmethods known in the art as necessary to quantify cellular uptakeefficiency.

Cellular Uptake Assay.

The various bicyclic peptides (cyclic CPP+peptidyl cargo) synthesizedaccording to Scheme 2 were assayed for cellular uptake efficiency usingflow cytometry and compared to cellular uptake efficiency of linearpeptidyl cargo (without the cyclic CPP).

All measurements were performed in triplicates and in the presence of10% fetal bovine serum (FBS). Attachment of a cargo peptide (e.g., anegatively charged peptide such as the Keap1 peptide) increases thecellular uptake (relative to CPP12).

Serum Stability.

The serum stability of the bicyclic peptides (cyclic CPP+peptidyl cargo)synthesized according to Scheme 2 were assayed and compared to cellularuptake efficiency of linear peptidyl cargo (without the cyclic CPP).Unconjugated peptides and CPP12-peptide conjugates were incubated in 25%human serum for varying periods of time (min) and the remaining intactpeptide was quantitated by analytical HPLC. Specifically, the serumstability of a bicyclic peptide comprising a cyclic CPP12 (FfΦRrRr)conjugated to a peptidyl inhibitor against the MDM2-p53 interaction(PMI) (FIG. 17 ), or conjugated to a peptidyl inhibitor against theKeap1-Nrf2 interaction (FIG. 18 ), or conjugated to a peptidyl inhibitoragainst Pin-1 (P1) (FIG. 19 ), compared serum stability of therespective linear peptidyl inhibitors.

Conjugation with CPP12 (via cyclization) greatly increases serumstability of all peptides.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A bicyclic peptide comprising a first cyclicpeptide and a second cyclic peptide, the bicyclic peptide having one ofthe following structures:

or a pharmaceutically acceptable salt thereof, wherein: the first cyclicpeptide comprises AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹, eachindependently an amino acid, wherein at least two amino acids arearginine and at least two amino acids independently comprise ahydrophobic side chain; each m, n, p, and q is independently selectedfrom 0 and 1; each d is independently 1 or 2; the second cyclic peptidecomprises a peptidyl ligand (X_(n)); R¹ is OH, OR², NHR²; and R² isalkyl, aryl, heteroaryl, amino acid, peptide sequence of 2 to 20 aminoacids, detectable moiety, or solid support.
 2. The bicyclic peptide ofclaim 1, wherein the at least two amino acid which independentlycomprise a hydrophobic side chain are glycine, phenylglycine, alanine,valine, leucine, isoleucine, norleucine, phenylalanine, tryptophan,naphthylalanine, proline, or combinations thereof, wherein the aromaticside chain on phenylglycine, phenylalanine, tryptophan, ornaphthylalanine are each optionally substituted with a halogen.
 3. Thebicyclic peptide of claim 2, wherein the at least two amino acid whichindependently comprise a hydrophobic side chain are phenylalanine,naphthylalanine, or combinations thereof.
 4. The bicyclic peptide ofclaim 1, wherein the at least two amino acids which independentlycomprise a hydrophobic residue are consecutive amino acids.
 5. Thebicyclic peptide of claim 1, wherein: AA¹ is L-arginine; AA² isL-arginine; AA³ is L-arginine; AA⁴ is L-naphthylalanine; and AA⁵ isL-phenylalanine.
 6. The bicyclic peptide of claim 1, wherein: AA¹ isL-phenylalanine; AA² is L-naphthylalanine; AA³ is L-arginine; AA⁴ isL-arginine; AA⁵ is L-arginine; and m is 1 and AA⁶ is L-arginine.
 7. Thebicyclic peptide of claim 1, wherein: AA¹ is L-arginine; AA² isL-arginine; AA³ is L-arginine; AA⁴ is L-arginine; AA⁵ isL-naphthylalanine; m is 1 and AA⁶ is L-phenylalanine.
 8. The bicyclicpeptide of claim 1, wherein at least three consecutive amino acids havealternating chirality.
 9. The bicyclic peptide of claim 8, wherein theat least three consecutive amino acids having alternating chirality arearginines.
 10. The bicyclic peptide of claim 9, wherein: AA¹ isD-phenylalanine; AA² is L-naphthylalanine; AA³ is L-arginine; AA⁴ isD-arginine; AA⁵ is L-arginine; and m is 1 and AA⁶ is D-arginine.
 11. Thebicyclic peptide of claim 9, wherein: AA¹ is D-phenylalanine; AA² isL-naphthylalanine; AA³ is L-arginine; AA⁴ is D-arginine; AA⁵ isL-arginine; m and n are each 1, and AA⁶ is D-arginine and AA⁷ isL-arginine.
 12. The bicyclic peptide of claim 9, wherein: AA¹ isL-phenylalanine; AA² is D-phenylalanine; AA³ is L-naphthylalanine; AA⁴is L-arginine; AA⁵ is D-arginine; and m and n are each 1, and AA⁶ isL-arginine and AA⁷ is D-arginine.
 13. The bicyclic peptide of claim 1,wherein X_(n) inhibits at least one protein-protein interaction.
 14. Thebicyclic peptide of claim 13, wherein the protein-protein interaction isan interaction between a IκB-kinase (IKK) complex and a regulatoryprotein NF-κB essential modifier (NEMO).
 15. The bicyclic peptide ofclaim 13, wherein X_(n) is an inhibitor against Ras, PTP1 B, Pin 1, Grb2SH2, or MDM2.
 16. The bicyclic peptide of claim 1, wherein X_(n) is awild-type peptidyl ligand or a peptide mimetic.
 17. The bicyclic peptideof claim 1, whereinAA¹-AA²-AA³-AA⁴-AA⁵-(AA⁶)_(m)-(AA⁷)_(n)-(AA⁸)_(p)-(AA⁹)_(q) is selectedfrom any one of SEQ ID NOS: 64-99, 102-118, and 120-146.
 18. A methodfor delivering a therapeutic agent to cytoplasm of a cell, comprisingadministering a compound comprising the bicyclic peptide of claim 1.